Wise/sost nucleic acid sequences and amino acid sequences

ABSTRACT

The present invention relates to nucleic acid sequences and amino acid sequences which influence bone deposition, the Wnt pathway, ocular development, tooth development, and may bind to LRP. The nucleic acid sequence and polypeptides include Wise and Sost as well as a family of molecules which express a cysteine knot polypeptide. Additionally, the present invention relates to various molecular tools derived from the nucleic acids and polypeptides including vectors, transfected host cells, monochronal antibodies, Fab fragments, and methods for impacting the pathways.

This application is a non-provisional patent application based on U.S.Provisional Patent Application Ser. No. 60/388,970, filed Jun. 14, 2002.

FIELD OF INVENTION

The present invention relates to Wise and Sost nucleic acid sequencesand related amino acid sequences that can be used to influence bonedeposition, the Wnt pathway, tooth development, and ocular development.In particular, the present invention also relates to nucleic acidsequences and amino acid sequences that optionally regulate or suppressbone deposition. The present invention relates to a family of nucleicacid molecules which expresses a family of amino acid sequences, some ofwhich are characterized by a cysteine knot, such as the Wise and Sostproteins. The present invention also relates to resultant molecularbiology tools derived from Wise or Sost, including plasmids, transfectedhost cells, antibodies, transfected host organisms, and knockoutorganisms. Finally, the present invention relates to the interactionbetween Wise or Sost and LRP.

BACKGROUND OF INVENTION

To activate and study the Wnt pathway, a wide range of materials andinformation has been used. Various model organisms explained below areused because of differing developmental characteristics associated withthe organisms. Because frogs and mice are exemplary of the organisms ofstudy, they are explained in greater detail below. As will be seen,frogs and mice were used in many of the Examples contained herein.Additionally, various genes and the Wnt pathway are explained.

Background of the Frog.

Frogs, in particular Xenopus, are excellent model organisms for testingembryonic development. Two species of Xenopus are commonly used fortesting, Xenopus laevis and Xenopus tropicalis. Both Xenopus species arenatives of Africa. Xenopus laevis has been used for many years toinvestigate the early period of embryonic development. Embryos developrapidly after fertilization, and a tadpole with a fully functional setof organs forms within a couple of days. Thus, experiments can beconducted on the embryos directly following fertilization. The embryoscan develop in a simple saline solution over a few days. The tadpolesare then examined to determine if the experimental intervention had anyobservable effect. The role of genes in development can be assayed byinjecting a tiny amount of any messenger RNA (mRNA) encoding the gene ofinterest into an early embryo, then once again allowing the embryo togrow into a tadpole.

The Xenopus embryo has long served as a major model for the study ofembryonic development because of its numerous advantages, includingexternal development, large size, identifiable blastomeres, and itsability to withstand extensive surgical intervention and culture invitro. These advantages enable extensive investigation of the earliestembryonic patterning events. In fact, much of the current understandingof early embryonic development derives from experiments performed in theXenopus embryo.

More particular to the frog, the earliest events of all animal embryosare controlled by mRNAs that are deposited in the egg by the mother.These maternal mRNAs control the embryonic processes that occur prior tothe transcription of the embryonic genome. These processes can best beexamined in Xenopus because, in these embryos, they occur during anespecially long period of time, and because they occur while the embryois developing externally. Such features have resulted in a detailedcellular and molecular understanding of early patterning events,including a comprehensive view of the role of specific extracellulargrowth factors, cell surface receptors, and intracellular signalingpathway components. These events include the patterning of the basicbody plan, the determination of cell fate, and the early patterning ofmajor organs, including the digestive system, circulatory system, andnervous system. In addition, many of the factors originally identifiedin Xenopus have been subsequently shown to control numerous laterdevelopmental events, as well as other critical biological processes,and oncogenesis. Finally, Xenopus is a major contributor tounderstanding cell biological and biochemical processes, includingchromosome replication; chromatin, cytoskeleton, and nuclear assembly;cell cycle progression; and, intracellular signaling. Thus, Xenopus isideally suited for studying early embryonic patterning and cell fatedetermination, later development, and organogenesis, oncogenesis, andcell biological and biochemical processes.

Background of A-P Patterning.

The mechanisms that generate regional differences along the anteriorposterior (A-P) axis of the vertebrate nervous system play an importantrole in pattern formation during development. The classicalactivation/transformation model proposed by Nieuwkoop suggests that aninitial signal induces neural tissue of anterior type and then a secondtransforming signal differentially acts on it to convert cells to a moreposterior character (Nieuwkoop, 1952; Slack and Tannahill, 1992). Thistransformer or “posteriorizing factor(s)” thus modifies a ground stateto generate the full spectrum of neural structures along the A-P axis.However, patterning of the anterior region is clearly more complicatedthan a simple default state of neural induction. This is highlighted bythe presence of local inductive centers, such as the anterior visceralendoderm and the isthmus, which are essential for anterior neuraldevelopment. Hence, models for a coordinated mechanism of A-P patterningin the nervous system need to integrate the influence of local signalson rostral brain patterning, with the influence of posteriorizingfactors that work more generally on the hindbrain and spinal cord.

Analysis of posteriorizing signals in neural patterning is complicatedby the tissue interactions and dynamic morphogenetic movements whichoccur during gastrulation. Xenopus animal caps provide a simplifiedsystem for studying patterning events separately from morphogeneticmovements. Animal caps alone form epidermis in culture, but when treatedwith antagonists of Bone Morphogenic Protein (BMP) signaling, such asNoggin, Chordin, Follistatin, or truncated BMP receptors, they adopt ananterior neural fate. Using these molecules as neural inducers,experimental studies in animal caps have provided evidence thatfibroblast growth factor (FGF), retinoic acid (RA), and Wnt (Winglessand iNT-1) signaling pathways influence A-P patterning by inducingposterior characters. Wnt is also known as the canonical Wnt pathway andthe Wnt planar polarity pathway. Thus, Xenopus embryo assays andexperiments in other vertebrates provide more evidence that RA, FGF, andWnt pathways influence A-P patterning. It is desired to betterunderstand the relative roles of these biochemical cascades, the degreeto which they are used at any particular axial level, and how they areintegrated in organizing normal A-P patterning.

Mesoderm plays an important early role in A-P patterning of neuraltissue. Mesoderm is the middle layer of embryonic cells between theectoderm and endoderm in triploblastic animals, and forms muscle,connective tissue, blood, lymphoid tissue, the linings of all the bodycavities, the serosa of the viscera, the mesenteries, and the epitheliaof the blood vessels, lymphatics, kidney, ureter, gonads, genital ducts,and suprarenal cortex. Experiments in Xenopus have shown that planarsignals within the neuroectoderm and vertical signals from theunderlying mesoderm work in concert to control regional identity of thenervous system. While early A-P specification of the nervous systemoccurs during gastrulation, it is not irreversibly committed to aparticular identity. Grafting experiments in several species revealplasticity in regional character and show that mesoderm is still playinga role at later stages. For example, analysis of the Hoxb4 gene hasshown that its expression pattern is established through interactivesignaling between the neural tube and the surrounding mesoderm.Furthermore, somites and paraxial mesoderm are sufficient to re-programHox expression in the neural tube to a more posterior character whengrafted ectopically. The ability of mesoderm to regulate regionalcharacter from early gastrula stages and to program motor neuronsub-type identities further emphasizes the importance of mesoderm andits signaling in patterning the developing nervous system.

The study of A-P patterning and focus on the mesoderm is of particularimportance in the present invention because such patterning impacts bonedevelopment in an embryo. Pathways which control A-P patterning oftenimpact bone development.

As such, it is desired to better understand the process ofposteriorization. The identification of new factors that can modulateexisting pathways, such as Wnts, FGF, and RA, or which represent novelsignaling inputs will be beneficial to understanding how A-P patterningis coordinated. In particular, it is desired to understand how the Wntpathway is activated and controlled. Xenopus has been used to study A-Ppatterning, that, in turn, is apparently impacted by the Wnt pathway.Xenopus can also be used to study activators or inhibitors of the Wntpathway.

Background of Mouse Model.

Mice are also excellent model organisms for testing embryonicdevelopment. Mice and humans possess similar genes, mice show manyclinical symptoms of human disease, and powerful techniques areavailable for genetic alterations of the mouse genome. All of thesefactors make mice excellent experimental models for testing newtherapies. Mice share many fundamental biological processes with humanstherefore, mice are considered to be one of the most significantlaboratory models for human disease and genetic mutations. Researchregarding human biological processes and genetic diseases can be greatlyenhanced by studying the mouse model for similar biological processesand diseases.

Mice have been a preferred experimental model for a number of years dueto their small size, short life span, and the female's ability toproduce a litter within two months after her birth. These factors allowresearchers to follow a given disease process from beginning to endwithin a short time frame. For these various reasons, mouse models arepreferred for testing new drug therapies, designing novel therapies, andstudying genetic diseases potentially also affecting humans.

Genes can be inserted into a fertilized mouse egg by several methodsincluding physical injection. The gene is first attached to a promoterand then is injected into the fertilized egg. The fertilized egg isimplanted into a female mouse and the embryo is allowed to develop to aspecified given stage for study. Once embryos reach the desired stage ofdevelopment, they can be harvested and tested to determine experimentalresults. Alternatively, embryos can be allowed to develop into full-termpups prior to being harvested to determine the results of theexperiment.

Because mice are phylogenetically closely related to humans with regardsto biological processes and diseases, and because of the rapidity ofmouse embryological development, they are considered to be an excellentanimal model for the study of human development, biological processes,and disease.

Background of Wnt.

Wnt proteins form a family of highly conserved secreted signalingmolecules that regulate cell-to-cell interactions during embryogenesis.Wnt genes and Wnt signaling are also implicated in aberrant cancer cellregulation. Insights into the mechanisms of Wnt action have emerged fromseveral systems: genetics in Drosophila and Caenorhabditis elegans (C.elegans); and, biochemistry in cell culture and ectopic gene expressionin Xenopus embryos. Many Wnt genes in the mouse have been mutated,leading to very specific developmental defects. As currently understood,Wnt proteins bind to receptors of the Frizzled family on the cellsurface. Through several cytoplasmic relay components, the signal istransduced to β-catenin, which then enters the nucleus and forms acomplex with TCF or LEF to activate transcription of Wnt target genes.The extracellular Wnt ligand binds the transmembrane receptor Frizzled(Fz), which activates the cytoplasmic phosphoprotein Dishevelled (Dsh).Activated Dsh inhibits GSK3 β-mediated degradation of β-catenin.β-catenin protein, therefore, accumulates and, in association withtranscription factors (TCF-3, TCF-4, LEF), regulates gene transcriptionin the cell nucleus.

Wnt-proteins, secreted glycoproteins, serve as important signalingmolecules during development of invertebrates and vertebrates. They havebeen shown to play crucial roles in such diverse processes as cancer,organogenesis, and pattern formation. To date, 19 Wnt genes have beenisolated in higher vertebrates, 7 have been found in the genome ofDrosophila, and 5 in the C. elegans genome. Wnt genes are defined bytheir sequence similarity to the founding members, Wnt-1 in the mouse(originally called iNT-1) and wingless (Wg) in Drosophila. The geneticanalysis of the Wg signaling pathway in Drosophila has led to theidentification of many downstream components, which have been shown tobe functionally conserved in other organisms. Wg/Wnt-proteins arethought to signal through seven-transmembrane receptors encoded by theFrizzled (Fz) gene family to regulate the stability of an effectorprotein known as armadillo (Arm) in flies or β-catenin (β-cat) invertebrates, which eventually leads to the activation of target genesthrough a complex of Arm/β-cat, with DNA-binding transcription factorsof the TCF/LEF family. This pathway is referred to as the canonicalWnt-pathway.

In recent years, evidence has been provided that Wnt signaling in thechick is involved in a variety of processes associated withskeletogenesis, such as chondrogenesis and joint development.Previously, it has been shown that there are at least three Wnt genes,Wnt-4, Wnt-5a, and Wnt-5b, as well as components of the canonical Wntsignaling pathway, expressed in chondrogenic regions, and that there isa fourth Wnt gene, Wnt-14, which is expressed early in the joint formingregion (FIG. 1D). Wnt-4 is also expressed in regions of the joint,however, its expression is restricted to cells in the periphery of thejoint interzone (FIG. 1C). Wnt-5a expression is restricted to cells in aregion of the perichondrium which will develop into the periosteum (FIG.1A), while the closely related Wnt-5b gene is expressed in asub-population of prehypertrophic chondrocytes, as well as cells of theouter layer of the perichondrium (FIG. 1B).

Much of what is known about the functional role of Wnt signaling inearly vertebrate development comes from experiments with Xenopus.Maternally encoded components of the canonical Wnt signaling pathwayfunction to establish the endogenous dorsal axis. The sperm fertilizingthe egg triggers cortical rotation. Vesicles are moved towards thefuture dorsal side. A dorsal determinant, which is likely to beDishevelled, is transported with these vesicles. Dishevelled accumulateson the dorsal side and inhibits GSK3. β-catenin can therefore accumulateon the dorsal side and, together with XTCF-3, induce the expression ofsiamois, which regulates down-stream dorsal development.

As such, the Wnt signaling system is one of only a limited number ofsignaling systems used during embryonic development to pattern theultimate resultant morphological physical body construction plan.Clearly, Wnt signaling is triggered at several discrete time pointsduring development, both at different developmental stages and withindifferent tissues (see Table below).

TABLE 1 Gene Expression Function XWnt-1 anterior neural mid-/hindbrainboundary XWnt-2 (=XWnt-2B) neural and heart not known XWnt-3-A posteriorneural neural anteroposterior patterning XWnt-4 neural, kidney kidneymorphogenesis (pronephros) XWnt-5A ectoderm not known XWnt-8 ventralmesoderm mesodermal patterning XWnt-8b forebrain not known XWnt-11dorsal marginal zone gastrulation movements

Early Xenopus development provides an excellent model system forstudying the general questions of tissue-specific response to Wntsignaling. Before the onset of zygotic transcription at the Mid-BlastulaTransition (MBT) phase, the Wnt pathway functions to establish thedorsal body axis. Only an hour or two later, after MBT, XWnt-8 functionsto promote ventral and lateral mesoderm. These strict stage-specificresponses to Wnt signaling could conceivably be induced by differentialuse of the canonical and alternative Wnt signal transduction pathways.

It is further known to those of skill in the art that Wnt genes areactive in osteoblast cells. Wnt regulates bone deposition in embryos andin mature individuals. It has been found that Wnt signals impact thedorsal-ventral pattern in early Xenopus embryo. In late embryos, Wntcauses anterior-posterior patterning of the neural tissue, neural crestformation, and organogenesis. As such, it is desired to havecompositions and methods for controlling Wnt signaling. Suchcompositions and methods would have impact on embryonic developmentalprocesses such as anterior-posterior patterning and on bone deposition.

Background of Sost.

Sost is believed to be a Bone Morphogenic Protein (BMP) antagonist.Mutations in the human Sost gene on human chromosome 17 can result insclerosteosis, which is an autosomal recessive sclerosing bone dysplasiacharacterized by progressive skeletal overgrowth. A high incidence ofthe bone dysplasia disorder, occurring as a result of a founder effectin affected individuals has been reported in the Afrikaner population ofSouth Africa, where a majority of individuals are affected by thedisorder. Homozygosity mapping in Afrikaner families, along withanalysis of historical recombinants, localized sclerosteosis to aninterval of ˜2 cm. between the loci D17S1787 and D17S930 on chromosome17q12-q21. Affected Afrikaners carry a nonsense mutation near the aminoterminus of the encoded protein, whereas an unrelated affected person ofSenegalese origin carries a splicing mutation within the single intronof the gene. The Sost gene encodes a protein that shares structural andfunctional similarity with a class of cysteine knot-containing factors,including dan, cerberus, gremlin, and caronte. The specific andprogressive effect on bone formation observed in individuals affectedwith sclerosteosis suggests that the Sost gene encodes a regulator ofbone homeostasis.

As such, evidence is provided herein that the deficiency of the Sostgene product, a novel secreted protein expressed in osteoblasts, leadsto the increased bone density in sclerosteosis. The two nonsensemutations, and the splice site mutation, are loss-of-function mutations.Previously, the precise function and working of Sost was believedunknown, an inhibitory effect on bone formation can be proposed sincepathophysiological analysis indicated that sclerosteosis is primarily adisorder of increased formation of normal bone. While it is known thatSost impacts bone formation, it is desired herein to better delineatethe mechanism of action and pathway of Sost's bone deposition activity.Previously, it has been hypothesized that Sost affected BMP rather thanthe Wnt pathway. Previous to our described invention herein, it was notknown that Sost reacted with Wnt pathway elements. The Sost-Wnt pathwayinteraction can be alternatively direct or indirect in nature.

Background of LRP6.

LRP genes encode the low-density lipoprotein (LDL)-receptor-relatedproteins, LRP5 and LRP6. Human LRP5 and LRP6 share 71% amino-acididentity, and together with Arrow, form a distinct subgroup of the LRPfamily. Arrow, LRP5, and LRP6 each contain an extracellular domain withepidermal growth factor (EGF) repeats and low-density lipoproteinreceptor (LDLR) repeats, followed by a transmembrane region and acytoplasmic domain lacking recognizable catalytic motifs. An LRP6mutation in mice results in pleiotropic defects recapitulating some, butnot all, of the Wnt mutant phenotype. LRP5/LRP6 involvement in Wntsignaling and LRP function in Wnt-induced axis Xenopus embryos have beenpreviously studied.

LRPs and Arrow in Drosophila are long single-pass transmembraneproteins. These proteins are of interest because they interact with andaffect Wnt signaling. Arrow is genetically required for Wingless (Wg)signaling (Webril, 2000) and mouse LRP mutations are similar inphenotype to Wnt mutants (Pinson, 2000). In Xenopus, over-expression ofLRP can activate Wnt signaling (Tamai, 2000). There is evidence thatWnts can bind directly to the extra-cellular domain of LRP and form aternary complex with the Frizzled receptor (Tamai, 2000). Also, thecytoplasmic domain of LRP can interact with Axin (Mao, 2001). Thus,LRP/Arrow appear to be important to understanding Wnt.

As stated, the Frizzled (Fz) family of serpentine receptors function asWnt receptors, but how Fz proteins transduce signaling is notunderstood. In Drosophila, Arrow phenocopies the Wingless (DWnt-1)phenotype, and encodes a transmembrane protein that is homologous to twomembers of the mammalian low-density lipoprotein receptor (LDLR)-relatedprotein (LRP) family, LRP5 and LRP6. It is reported that LRP6 functionsas a co-receptor for Wnt signal transduction. In Xenopus embryos, LRP6activated Wnt-Fz signaling, and induced Wnt responsive genes, dorsalaxis duplication, and neural crest formation. An LRP6 mutant lacking thecarboxyl intracellular domain blocked signaling by Wnt or Wnt-Fz, butnot by Dishevelled or β-catenin, and inhibited neural crest development.The extracellular domain of LRP6 bound Wnt-1 and associated with Fz in aWnt-dependent manner. This indicates that LRP6 is likely to be acomponent of the Wnt receptor complex.

Further, Wnt/β-catenin signaling induces dorsal axis formation throughactivation of immediate, early responsive genes, including nodal-related3 (Xnr3) and Siamois (Sia). It has been shown that in two developmentalprocesses dependent on the Wnt pathway in Xenopus—secondary axis andneural crest formation—LRP6 activates, but a dominant-negative LRP6inhibits, Wnt signaling, providing compelling evidence that LRP6 iscritical in Wnt signal transduction. LRP6 functions upstream of Dsh inWnt-responding cells, synergizes with either Wnt or Fz, and importantly,is able to bind Wnt-1 and to associate with Fz in a Wnt-dependentmanner. The simplest interpretation of these findings is that LRP6 is acomponent of the Wnt-Fz receptor complex.

Genetic studies of Arrow in Drosophila and LRP6 in mice strongly supportthis hypothesis. Data also indicates the possibility that Wnt-inducedformation of the Fz-LRP6 complex assembles LRP6, Fz and their associatedproteins, thereby initiating cytoplasmic signaling. Consistent with thisnotion, Wnt signal transduction requires intracellular regions of bothFz and LRP6, which harbor candidate protein-protein interaction motifs.Notably, Arrow does not exhibit Fz planar polarity phenotype, implyingthat Arrow-LRP6 may specify Wnt-Fz signaling towards the β-cateninpathway. How Fz, LRP6, and proteoglycan molecules, such as Dally,interact to mediate Wnt recognition/specificity, and signal transductionremains to be elucidated. Thus, it is understood that LRP interacts withWnt. The present invention is designed and characterized to control LRPbinding to Wnt and Fz, and, more particularly, to control LRP upstream.

Background of LRP5.

In humans, low peak bone mass is a recognized significant risk factorfor osteoporosis. It has been reported that LRP5, encoding theLDLR-related protein 5, affects bone mass accrual during growth.Mutations in LRP5 cause the autosomal recessive disorderosteoporosis-pseudoglioma syndrome (OPPG). OPPG is an autosomalrecessive disease, characterized by severe osteoporosis due to decreasedbone formation and pseudoglioma resulting from failed regression ofprimary vitreal vasculature. Y. Gong, et al. (2001). Gain of genefunction leads to high bone mass (HBM) phenotype as an autosomaldominant trait, whereas loss of function leads to osteoporosis.

It has been found that OPPG carriers have reduced bone mass whencompared to age- and gender-matched controls. LRP5 expression byosteoblasts in situ has been demonstrated and LRP-5 has been shown toreduce bone thickness in mouse calvarial explant cultures. These dataindicate that Wnt-mediated signaling via LRP5 affects bone accrualduring growth and is important for the establishment of peak bone mass.

In mice, it has been found that LRP5 participates in bone formation andbone mass. Null mutation of LRP5 causes post-natal bone loss, resultingfrom decreased bone formation and osteoblast proliferation, independentof Runx2. M. Kato, et al. (2002). In contrast, transgenic miceexpressing LRP5 with the HBM mutation G171V exhibit increased boneformation and bone mass, without skeletal developmental abnormalities.F. Bex, et al. (2002).

LRP5 appears to interact with the Wnt pathway since LRP5 with the HBMmutation prevents inhibition of Wnt signaling by Dikkopf-1. L. M. Boyden(2002); A. M. Zorn (2001). There is murine hybridization and microarrayevidence that indicates Wnt signaling is involved in bone fracturerepair. M. Hadjiargyrou (2002). Six additional mutations in LRP5,located in the amino-terminal domain near G171, have been identified.These mutations cause increased bone density, particularly in corticalbone. L. Van Wesenbeeck (2003).

Background of β-Catenin.

β-catenin reports demonstrate its accumulation opposite the sperm entrypoint by the end of the first cell cycle. β-catenin continues toaccumulate in dorsal (i.e., opposite the sperm entry point) but notventral cytoplasm through the early cleavage stages. By the 16- to32-cell stages, it accumulates in dorsal but not ventral nuclei.Remarkably, the pattern of dorsal accumulation of β-catenin closelyparallels the ability of transplanted dorsal cells to induce an axiswhen implanted into host embryos. Thus, β-catenin is the first signalingmolecule to show a dorso-ventral polarity in the early embryo. Combinedwith the loss-of-function data from Heasman et al., it is now clear thatwhen fertilization elicits a cortical rotation, and displacement ofmaterial and organelles to the future dorsal side, it leads to adorso-ventral asymmetry in β-catenin, which is required for axisformation.

Brannon et al. show that the HMG Box factor XTCF-3 directly binds thesiamois promoter. In the absence of β-catenin, XTCF-3 inhibits geneexpression. However, on the dorsal side of the embryo, β-catenin bindsthe XTCF-3, and, thus, activates the gene. This is notable becausesiamois is a homeobox gene likely playing a major role in specifyingformation of Spemann's Organizer. Therefore, a dorso-ventral differencein β-catenin forms within an hour or two of fertilization, directlyregulating a key homeobox gene in the blastula, thus contributing toformation of Spemann's Organizer on the dorsal side of the gastrula.

β-catenin not only impacts development, but it influences bonedevelopment in adults. Regulation of osteoblasts results fromaccumulation of β-catenin in the cell. It is desired to have methods andcompositions for controlling bone deposition. It is known that the Wntpathway controls accumulation of β-catenin, which regulates osteoblastexpression. It is desired to control and inhibit osteoblast regulationby preventing Wnt pathway activation. For this reason, the presentinvention includes nucleic acid molecules and amino acid sequences forcontrolling Wnt.

SUMMARY OF INVENTION

The present invention relates to Wise nucleic acid sequences and aminoacid sequences, Sost nucleic acid sequences and amino acid sequences,and LRP nucleic acid sequences and amino acid sequences. Additionally,the present invention relates to control over the influencing of bonedeposition, ocular development, tooth development, and the Wnt pathwayusing the above nucleic acid sequences and amino sequences.Additionally, the present invention relates to molecular tools developedfrom the nucleic acids and polypeptides including vectors, transfectedhost cells, transfected organisms, knockout organisms, antibodies,hybridomas cells, Fab fragments, and homologous nucleic acid sequencesand polypeptides. Mutants of the Wise, Sost, and LRP nucleic acidsequences and polypeptides are contemplated herein and are used toinfluence the pathways. The Wise and Sost nucleic acid sequences aregenerally about 70% homologous. Related to this are cysteine knotpolypeptides which bind to LRPs as well as a variety of polypeptides.There is a family of nucleic acid sequences and polypeptides expressedtherefrom, which are related to the Wise and Sost sequences. The hostcells that can be treated with the mutants of the present inventioninclude insects, amphibian, and mammalian cells.

Nucleic acid sequences, and the resultant polypeptides, are members of afamily of isolated nucleic acid molecules which influence one of thefollowing: tooth development, Wnt pathway activation, bone deposition,or ocular development is contemplated herein. The family includes avariety of nucleic acid molecules including NDP, DAN, Caronte, PDGF,Wise, Sost, Cereberus, Gremlin, CTGF, Soggy, DKK1, Cyr61, DKK2, DKK3,DKK4, NOV, Mucin, Slit, OOH, Wisp, and CCN. Related to this are the LRPfamily of molecules which also influence these various pathways. Inparticular, LRP 1, 2, 5, and 6. As such, the family that expresses acysteine knot polypeptide binds to one of the LRPs. The various nucleicacids are specifically listed in the Sequence ID listing includedherewith. Related to this are degenerate variants of the nucleic acidmolecules. As mentioned, the family of nucleic acid molecules typicallyexpresses a polypeptide that includes a cysteine knot protein, with thecysteine knot protein including eight cysteine residues. However,variations of the cysteine knot protein are available for use. As such,any nucleic acid sequence which impacts the previously mentionedpathways and expresses a cysteine knot protein is believed related tothe present family of nucleic acid sequences. It is known that Exon 2 ofthe Wise nucleic acid sequence (SEQ. ID. NO. 128) expresses a desiredcysteine knot protein. As such, oligonucleotide fragments which are 70%homologous to Wise Exon 2 are believed to be potentially related to thepresent family of nucleic acid molecules.

Mutant versions of the above nucleic acid molecules can result inincreased bone deposition, as well as tooth development and oculardevelopment. Additionally, the mutants will influence with Wnt pathwayactivation. As such, mutant versions of the nucleic acid molecules ofthe present invention are known to impact the mentioned pathways in avariety of ways. The present invention resultingly relates to any mutantversion of the listed nucleic acid sequences. The mutants can begenerated via point, frame shift, deletion, or loss of functionmutations. Loss of function mutations can be achieved by placing a stopcodon near the beginning of the selected nucleic acid sequences, whichwould include before or after the start of the sequence. For example, astop codon can be placed just after the start of Exon 1 of the Wisenucleic acid sequence. During translation the stop codon will preventtranslation of the Wise Exons and therefore the polypeptide will not beexpressed. Other available mutants include antisense RNAs, morpholinos,antisense oligonucleotides, mRNAs translated from the selected nucleicacid sequences, and RNAi complementary to the nucleic acids sequences.

As discussed herein, nucleic acid sequences and nucleic acid moleculeswill be used interchangeably. The isolated nucleic acid sequencesinclude gDNAs, cDNAs, and a variety of other nucleic acid sequencefragments. It is contemplated that any of a variety of nucleic acidsequences can be used herein including genes, mRNA, cDNA, gDNA, tRNA,oligonucleotides, polynucleotides, and nucleic acid sequence fragments.As such, any nucleic acid sequence which expresses a polypeptide thatinfluences either tooth development, Wnt pathway activation, bonedeposition, or ocular development is contemplated as part of the presentinvention, as well as mutant versions thereof. The nucleic acidsequences will include genes which are any hereditary unit that has anaffect on the phenotype of an organism and can be transcribed into mRNAswhich result in polypeptides, as well as rRNAs or tRNA molecules andregulatory genes. Also, alleles and mutant alleles are part of thedefinition of a gene as used herein.

Probes which hybridize to either mutant nucleic acid sequences or thenon-mutant nucleic acid sequences are part of the present invention. Theprobes will include any of a variety of labels and can be either cDNA orRNA probes. The probes can be used to form a kit or similar tool for usein detecting the presence or absence of a particular Wise, Sost, or LRPnucleic acid or polypeptide.

Vectors are formed from both the isolated nucleic acid sequences and themutant versions of the isolated nucleic acid sequences. The vectorsinclude expression, cloning, and viral vectors. Other available vectorsinclude fusion vectors, gene therapy vectors, two-hybrid vectors,reverse two-hybrid vectors, sequencing vectors, and cloning vectors.Also, prokaryotic and eukaryotic vectors may be used. Specificprokaryotic vectors that may be used in the present invention includepET, pET28, pcDNA3.11V5-His-TOPO, pCS2+, pcDNA II, pSL301, pSE280,pSE380, pSE420, pTrcHis, pRSET, pGEMEX-1, pGEMEX-2, pTrc99A, pKK223-3,pGEX, pEZZ18, pRIT2T, pMC1871, pKK233-2, pKK38801, and pProEx-HT.Specific eukaryotic vectors that may be used herein include pFastBac,pFastBac HT, pFastBac DUAL, pSFV, pTet-Splice, pEUK-C1, pPUR, pMAM,pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, YACneo, pSVK3, pSVL, pMSG,pCH110, pKK232-8, p3′SS, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3,pREP4, pCEP4, and pEBVHis. As mentioned, a variety of promoters may beused with the vector, as well as any of a variety of selectable markers.Available markers include antibiotic resistance genes, a tRNA gene,auxotrophic genes, toxic genes, phenotypic markers, colorimetricmarkers, antisence oligonucleotides, restriction endonuclease, enzymecleavage sites, protein binding sites, and immunoglobulin binding sites.Specific selectable markers available include LacZ, neo, Fc, DIG, Myc,and FLAG.

Any of a variety of host cells, including prokaryotic and eukaryoticcells, can be transfected with the vectors previously mentioned.Prokaryotic host cells include Gram-negative and Gram-positivebacteriums may be transfected with any of the variety of the vectorspreviously mentioned. Available bacteriums include Escherichia,Salmonella, Proteus, Clostridium, Klebsiella, Bacillus, Streptomyces,and Pseudomonas. A preferred. Gram-negative bacterium is Escherichiacoli. Eukaryotic vectors can be used to transfect eukaryotic host cellsincluding yeast, plant, fish, mammalian, human, mouse, frog, or insectcells. Specific host cells that can be transfected include ES, COS, HEK293, CHO, SaOS, osteosarcomas, KS483, MG-63, primary osteoblasts,osteoclasts, chrondocytes, and human or mammalian bone marrow stroma. Assuch, the present invention includes host cells transfected with any ofthe previously mentioned vectors.

It is specifically contemplated that mutant Wise nucleic acid sequencescan be used. The mutant Wise nucleic acid sequences will be mutatedversions of SEQ. ID. NO. 1-5, 126-128, 109, 96, and 97, as well ascomplementary mutant sequences thereof. Additionally, degeneratevariants of these sequences may also be used. Plasmids can be formedfrom these mutant Wise nucleic acid sequences, as well as transfectedhost cells. Additionally, mutant organisms can be formed from the mutantWise nucleic acid sequences, including Wise mutant mice. Sost and LRPcan also be mutantized and various related constructs can be formedtherefrom. Specific mutants to either Wise, Sost, or LRP can bedeveloped related to SEQ. ID. NO. 1-44, 96-103, 108, 110-113 and 126-128listed herein.

Amino acid sequences which influence at least one of the following,tooth development, Wnt pathway activation, bone deposition, or oculardevelopment are part of the present invention. Available amino acidsequences include those polypeptides or proteins expressed from thepreviously discussed nucleic acid molecules. In particular, Wise, Sost,and LRP polypeptides and amino acid sequences can be used herewith.Specifically available amino acid sequences include those listed in theSEQ. ID. NO. 45-95, 104-107, 109, 114-125. Isolated polypeptides thathave a cysteine knot formed from eight cysteine knot residues whichimpact the previously listed pathways are included herewith. Finally,amino acid sequences which are 70% homologous to Exon 2 polypeptides ofWise may be used herewith. When used herein amino acid sequences includeany of a variety of polypeptide and protein molecules.

Antibodies which bind to at least one of the previously mentioned aminoacid sequences are used herewith. The antibodies include monoclonal,polyclonal, recombinant, and antibody fragments. Any of a variety ofantibodies may be used that bind to either Wise, Sost, or LRP 1, 2, 5,or 6. The antibodies are designed to either bind to the selectedpolypeptide and prevent it from binding to its normal antigen.Conversely, the antibodies can be designed such that they attack anddestroy the chosen or selected polypeptides. For example, it ispreferred to bind either Wise or Sost with Wise or Sost antibody,respectively, whereby Wise or Sost is prevented from binding to LRP 5 or6. As such, it is desired to have antibodies that specifically bindWise, Sost, or LRP. Related to the antibodies are Fab fragments whichfunction the same way as the chosen antibodies. These anti-peptideantibodies will prevent binding by the selected amino acid sequence toan LRP for example. The antibodies can be directed to both mutant andnon-mutant versions of polypeptides expressed from the mutant ornon-mutant versions of the nucleic acid sequences.

Hybridomas can be formed which are used to produce the desiredantibodies. As such any of a variety of cells can be used to produceboth the polypeptides as well as the antibodies.

It is known that both Wise and Sost polypeptides bind to LRP 5 or 6polypeptides. As such, the present invention relates to a proteinmolecule formed from a Wise polypeptide bound to an LRP polypeptide.Additionally, the present invention relates to a Sost polypeptide boundto an LRP polypeptide.

Use of the isolated nucleic acid sequences or polypeptides canspecifically result in increased bone deposition, both in vivo and invitro. As such a variety of methods can be practiced which are designedto increase the bone deposition either in a selected cell or a selectedhost organism. One particular method includes isolating a nucleic acidsequence which can be either Wise, Sost, or LRP. The nucleic acidsequence then is used to form a cassette which includes a stop codon atthe beginning of the nucleic acid sequence. Preferably, the cassettewill include a marker and a promoter. The selected nucleic acid sequencecan be either a mutant or a non-mutant nucleic acid sequence, with thesequence selected dependent upon the desired outcome. The cassette isthen used to form a plasmid whereby any of a variety of plasmids, aspreviously mentioned, may be used. Once the plasmid is formed it is usedto transfect a host cell. Any of a variety of methods can be used totransfect a host cell including microinjection. The available host cellwill include a variety of prokaryotic and eukaryotic cells. Among theavailable cells are embryonic stem cells, blastomeres, and a variety ofother stem cells. Once the host cell is transfected the stop codon canbe activated to cause a loss of function mutation which results in aphenotypic change. Among the phenotypic changes are increased bonedeposition. The transfected host cells can also be used to transfect ahost cell organism such as a mouse. The cells are injected into anembryo with the embryo then allowed to develop or mature. Host cellsinclude insect, amphibian, and non-human mammal. Human cells can also betreated in vitro. Specific delivery of the nucleic acid sequence intothe host cell can be accomplished via microinjection, micro-vesselencapsulation, liposome encapsulation, and electroporation. Desired hostcells include osteoblasts, osteoclasts, and chrondocytes. Besidesattaching stop codons to the nucleic acid sequence in the plasmid, othermutantized versions may be used. In particular, an alternative to thestop codon are point mutations, frame shift mutations, and othermutations may be used to preclude accurate translation of thepolypeptide. This will resultingly achieve the same effect as a loss offunction mutation. In particular, antisense RNA vectors may be used inthe alternative.

Bone deposition can also be increased as an alternative method. Anucleic acid sequence can be selected, including Wise, Sost, or LRP. Anucleic acid sequence is then used to form a plasmid vector whereby thevector is used to transfect the host cell. The host cell will expressthe nucleic acid sequence to produce a polypeptide. Once a sufficientamount of polypeptide is produced it can be harvested for use inimmunizing a host organism. Available host organisms include mice, rats,goats, rabbits, and any of a variety of other organisms used to producepolypeptides. The immunized host organism will produce antibodies to thepolypeptide that was used to immunize the host. After a period of timethe antibodies may be isolated and separated from the host. Theantibodies can be used as is or can be further treated to produce Fabfragments or related small molecules. Regardless of the selected form ofthe antibody it can be combined with a carrier. Any of a variety ofcarriers are available for use including liposomes. The carrier antibodycombination is used to transfect a host cell. This can be done either invitro or in vivo. The antibody will bind to the selected targetpolypeptide and prevent activation of a selected pathway. This processcan also be used in association with the Wnt pathway, tooth developmentor ocular development.

Any of a variety of kits may be formed both to the polypeptides or thenucleic acid sequences of the previously mentioned constituents. Thekits can be used to detect the presence of a particular nucleic acidsequence or polypeptide or the absence of such composition.

BRIEF DESCRIPTION OF DRAWINGS

The application file contains at least one photograph executed in color.Copies of this patent application with color photographs will beprovided by the Office upon request and payment of the necessary fee.

FIG. 1 shows the isolation of Wise by Xenopus animal cap screening;

FIG. 1A shows an illustration of the screening procedure;

FIG. 1B shows the RT-PCR gel electrophoresis results of the first roundof screening;

FIG. 1C shows the RT-PCR analysis of injections using RNA from theisolated Wise clone;

FIG. 2 shows Wise as a conserved secreted protein;

FIG. 2A shows the alignment of the predicted amino acid sequence ofZebrafish, Xenopus, chick, mouse, and human Wise proteins. Shaded boxesrepresent identical amino acids between species; asterisks indicateresidues conserved in Drosophila Slit, and dots identify residuesconserved in Cef10. Circles mark conserved cysteine residues. Thearrowhead delineates the site of signal peptide cleavage predicted inthe chick clone;

FIG. 2B is a diagram showing alignment of conserved amino acids betweenWise, Slit, and Cef10 (a CCN family member). Filled ovals and red linesindicate cysteine residues in the Slit homology domain conserved in theCT domain of CCN family members but not in Wise or Slit. Dotted linesshow other conserved amino acids. Shaded boxes in Wise indicate threeblocks Δ1, Δ2, Δ3 deleted separately for functional analysis;

FIG. 2C shows Western blot detecting HA-tagged Wise protein secretedinto the medium following RNA injection into oocytes and controluninjected oocytes;

FIGS. 2D and 2E show the recombination between Noggin-expressing andWise-expressing animal caps assayed for expression of Krox 20 (FIG. 2D)or en2 (FIG. 2E). Wise induces a ring of En2 (en) expression or patchesof Krox20 staining in a non-cell autonomous manner in the Noggin cap. InFIG. 2D, the Noggin-injected cap was marked with FIDx, and in FIG. 2E,the Wise cap was marked with lacZ, as lineage tracers;

FIG. 3 shows the expression of Wise in chick and Xenopus embryos;

FIGS. 3A-3D shows the in situ hybridization of chick embryos. Wise isexpressed in the surface ectoderm from the level of presomitic mesodermto the posterior end at stage 10 (FIG. 3A). Higher transcript levels aredetected at stage 11 (FIG. 3B), which refine to a small posterior domainby stage 12 (FIG. 3C). In FIG. 3D, a section of FIG. 3A, in the vicinityof Hensen's node shows Wise transcripts confined to the surface ectoderm(se);

FIG. 3E shows the RNase protection of Xenopus embryos with stages notedabove each lane. Wise is first detected at an early gastrula stage, andthe expression persists into tadpole stages. ODC is a loading control;

FIGS. 3F and 3G shows the whole mount in situ hybridization to Xenopusembryos. At stage 15 (FIG. 3F), Wise is expressed in the surfaceectoderm at all anterior-posterior levels. The expression is strongestat the edge of the neural tube. At tadpole stages (FIG. 3G, stage 40),expression is localized in epibranchial placodes, lateral lines, andalong the dorsal fin;

FIG. 4 shows changes in neuronal markers after blastomere injection ofWise RNA and Wise antisense morpholino oligos;

FIGS. 4A-4L shows in situ hybridization with neural markers in stage16-21 Xenopus embryos following single blastomere injections of Wise RNA(FIGS. 4B, 4E, 4H, and 4K) at the 8-cell stage or antisense morpholinooligos (FIGS. 4C, 4F, 4I, and 4L) at the 4-cell stage. The left panels(FIGS. 4A, 4D, 4G, and 4J) indicate control embryos. In most embryos,lacZ (blue staining) was co-injected as a lineage tracer. Injected sidesare to the left. Probes were Sox3 (FIGS. 4A-4C), Ent (FIGS. 4D-4F),Krox20 (FIGS. 4G-4I), and Slug (FIGS. 4J-4L). In Wise RNA injectedembryos, the neural markers were generally displaced posteriorly.Ectopic induction of Krox20 and Slug can be seen in the forebrain region(FIGS. 4H and 4K). In embryos injected with antisense morpholino oligos,these markers were unchanged;

FIGS. 4M and 4N show the transverse sections at stage 16 afterblastomere injection of either Wise RNA (FIG. 4M) or Wise antisensemorpholino oligo (FIG. 4N). In FIG. 4M, the neural plate on the injectedside was greatly expanded, which is revealed by Sox3 staining (darkblue, *). Conversely, in the morpholino oligo-injected embryo (FIG. 4N),the surface ectoderm is thicker on the injected side (left, *) incomparison to the right control side;

FIG. 5 shows the anterior defects after blastomere injection of Wise RNAor morpholino oligo;

FIGS. 5A-5L shows in situ hybridization with the cement gland marker XCGat stage 16-20 (FIGS. 5A-5C) and morphological phenotypes of cementgland at stage 26-40 (FIGS. 5D-5F) or eye at stage 35-36 (FIGS. 5G-5I),in control embryos (FIGS. 5A, 5D, and 5G), Wise RNA injected embryos(5B, 5E, 5H), and morpholino oligo injected (FIGS. 5C, 5F, and 5I)embryos. Blue staining shows co-injected lacZ lineage tracer.Over-expression of Wise resulted in formation of larger cement glands(FIG. 5C). Eye formation is consistently blocked by injection of bothWise RNA (FIG. 5H) and the morpholino oligo (FIG. 5I);

FIG. 5J shows in vitro translation of Wise in the presence of the Wisemorpholino antisense oligo. Lane 1; translation of Wise protein withoutmorpholino oligo. Lanes 2-7; translation in the presence of the Wisemorpholino oligo at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM,10 μM, respectively. Lane 8; translation in the presence of controlmorpholino oligo at the concentration of 10 μM. Wise translation ispartially blocked at concentration of 1 μM, and completely blocked at 10μM;

FIG. 5K shows the rescue of the eye defect resulting from injection ofthe morpholino oligos by co-injection of Wise RNA;

FIGS. 5L-5N are the phenotypes of embryos following injection of Wisemorpholino oligos throughout the whole embryo. 5L shows the range ofcyclopic eye and short trunk phenotypes induced by the oligos incomparison to the control embryo (left). Section of control (FIG. 5M)and morpholino-injected (FIG. 5N) embryos at the level of eye. In theWise morpholino-injected embryos, eyes are positioned very close to theneural tube;

FIG. 6 shows that Wise requires components of the Wnt pathway for En2induction and stimulates translocation of β-catenin to the nucleus;

FIGS. 6A-6C show the RT-PCR of Noggin treated animal caps assayed forEn2 (en) induction. NCAM is used as a pan neural marker and Ef1a is aloading control. FIG. 6A shows the induction of En2 by Wnt8 or Wise RNAis blocked by dominant-negative (dn) Frizzled 8 ΔFz8. FIG. 6B shows theinduction of En2 is blocked by dn-Wnt8 (Wnt8), dn-Dishevelled ΔDsh(dd1),GSK3 and dn-Lef1 (LEFΔN). FIG. 6C shows the induction of En2 requiressignaling components of the canonical Wnt pathway but not the planarcell polarity (PCP) pathway. Wise-mediated En2 induction is abolished byΔDsh(dd1), a dominant negative form of Dishevelled for both pathways,and ΔDsh(DIX), which blocks the canonical pathway. ΔDsh(DEP) blocks thePCP pathway but has no effect on Wise induction of En. ΔDshΔNspecifically activates the PCP pathway but fails to induce En in theabsence of Wise, although full length dishevelled (Dsh) is able to doso;

FIGS. 6D-6G show the staining for sub-cellular localization ofendogenous β-catenin detected immunocytochemically in Xenopus animalcaps following RNA injection of: FIG. 6D, TCF3; 6E, Wnt8+TCF3; FIG. 6F,Wise+TCF3; and FIG. 6G, β-catenin+TCF3. Wnt8 (FIG. 6E) and Wise (FIG.6F) promoted accumulation of nuclear β-catenin;

FIG. 7 shows how Wise affects Wnt signaling;

FIGS. 7A-7C show the secondary axes induced by Wnt8 are blocked by Wise.Injection of Wnt8 RNA into a ventral vegetal blastomere of 4-8-cellstage embryos induces complete secondary axis formation (FIG. 7A).Co-injecting Wise blocks formation of Wnt8-induced secondary axis (FIG.7B), similar to the effect obtained by co-injection of a dominantnegative Dishevelled, ΔDsh(DIX) (FIG. 7C);

FIG. 7D shows Wise functions extracellularly to block induction ofsiamois and Xnr3 by the Wnt pathway in ventral marginal zones. Wiseblocks the ability of Wnt8 to induce Siamois and Xnr3, but does notinterfere with the ability of Dishevelled (Dsh) or β-catenin (β-cat) toinduce these markers;

FIGS. 7E and 7F show that Wise acts as Wnt inhibitor and induces headdevelopment in the incomplete secondary axis. When BMP signaling isblocked at the ventral marginal zone by injection of a truncated BMPreceptor (tBR), an incomplete secondary axis is formed (FIG. 7E).Co-injection of tBR and Wise induces a complete secondary axis with eyes(arrows) and cement gland (FIG. 7F);

FIGS. 7G-7I show that Wise blocks cell movements in Activin-treatedanimal caps. Control animal caps (FIG. 7G) undergo gastrulation-likemovements in the presence of Activin (FIG. 7H). In Wise injected animalcaps, elongation is blocked (FIG. 7I), but mesoderm induction occurs;

FIG. 8 shows that Wise interacts with the extracellular domain ofFrizzled 1, 3, 7, 8; and Western blotting of COS cell extracts fromcells transfected with epitope tagged protein variants. The relevantconstructs transfected into COS cells that were used to prepare eachextract are listed at the top of each column;

FIG. 8A shows that Frizzled binds to Wise, as well as to Wnt8;

FIG. 8B shows that Wnt8 interacts with Fz1 but not with Wise; and,

FIG. 8C shows that Wise interacts with Fz1, Fz3, Fz7, and Fz8;

FIGS. 8A-8C, in the top panels are controls showing that the Myc-taggedversions of each protein are present and recognized by the anti-Mycantibody. The middle panels are controls showing the presence ofproteins tagged with FLAG and recognized by the anti-FLAG antibody. Thebottom panels illustrate results of immuno-precipitation using theanti-Myc antibody and Western blotting with anti-FLAG to show proteininteractions. The antibodies used in each set of experiments areindicated at the left;

FIG. 9A is a schematic showing the gene structure for Wise and Sost;

FIG. 9A depicts the Neo-LacZ cassette insertion into Exon 1, which isseparated from Exon 2 by an intervening intron sequence;

FIG. 9B shows mouse Wise and Sost polypeptide sequences;

FIG. 9C shows Wise, Sost, and Hox A and B genes in chromosomes;

FIG. 9D illustrates the family tree map showing the relatedness of Wiseand Sost to other cysteine knot family members;

FIG. 9E shows the family of cysteine knot proteins and their alignedpolypeptide sequences;

FIG. 10 is a model of the Wise and SOST Exons, which express thecysteine knot structure. It depicts the 200 bp of Exon 1 and the 400 bpof Exon 2;

FIG. 11 shows the effects of Sost and Wise polypeptides on Xenopusembryonic development;

FIG. 11A shows that Wise and Sost defects lead to morphologicalabnormalities in Xenopus tadpoles;

FIG. 11B is a table showing Wise and Sost effects on Noggin and Wnt8expression in embryos;

FIG. 11C depicts Sost effects for Wnt8 and β-catenin with VMZ and DMZ;

FIG. 11D shows electrophoretic patterns for NCAM, En2 and EF1-α;

FIG. 11E shows electrophoretic patterns for Siamois, Xnr3, and EF1-α;

FIG. 12 shows the effect of the absence of a functional Wise polypeptidemolecule upon ophthalmic development in Wise knockout mice, whereinophthalmic and eye abnormalities developed in these mice.Immunodetection of Wise protein production in murine retinal regions wasused to determine the efficacy of induced Wise mutation;

FIG. 12A shows whole eye mounts containing retinas or sections that werestained with anti-Wise antibody and FITC-conjugated second antibody;

FIG. 12B shows that in wild type mice, anti-Wise reactivity was detectedas secreted Wise protein in the ganglion cell and optic fiber layers andin rods and cones. However, Wise mutant mice eyes lacked detectableanti-Wise peptide reactivity, indicating absence of Wise from tissues ofthese mutant mice. The Wise mutant mice appeared to have lost themajority of the optic nerve fibers and had increased rod and cone layersin the retina. Wise protein was found in the inner plexiform layer,ganglion cells and fibers, and in the rods and cone layer of a 2.5 monthmouse retina;

FIG. 12C shows immunofluorescence patterns for Wise, Pax6 and 2H3 intissue cross-sections;

FIG. 13 shows results of bone staining and bone mineral density (BMD)measurements;

FIG. 13A depicts hematoxylin and eosin (H&E) staining of cross-sectionsof bone tissue from 16 to 18 days post cortum (DPC) mice;

FIG. 13B illustrates the same bone regions as FIG. 13A; however, FIG.13B left shows staining with S-35 radiolabel attached to Sost RNAprobes, wherein Sost is located in osteoblasts in 16 to 18 DPC mice.FIG. 13B right also shows staining with anti-Wise peptide primaryantibody and FITC-conjugated secondary antibody, and localization ofWise in hypertrophic and prehypertrophic proliferating chondrocytes;

FIG. 13C shows graphical depictions of bone density measurements andtotal bone weight measurements, respectively. FIG. 13C left shows thatobservable significant differences in BMD measurements between Wisemutant and wild type mice at certain ages. FIG. 13C right depicts totalbone weight measurements. FIG. 13 generally shows both Sost and Wisegenes appear to affect bone cells. Sost is expressed in osteoblasts. Incontrast, Wise is expressed in periosteum, chondrocytes (proliferating,prehypertrophic and hypertrophic), but not in the growth plate;

FIG. 14A shows a bilateral view of two molars with developing tooth budson hemotoxylin and eosin staining of a tooth cross-section;

FIG. 14B shows a bilateral view of two molars with developing tooth budswith S-35 RNA probe-labeled Sost staining;

FIG. 14C shows a bilateral view of two molars with developing tooth budsstained with S-35 RNA probe-labeled Wise stain for purposes of detailingthe layers of the dental follicle surrounding the molar teeth;

FIG. 14D shows a molar tooth bud at a higher magnification with abilateral view of two molars on hematoxylin and eosin staining of atooth cross-section;

FIG. 14E shows a molar tooth bud at a higher magnification stained withS-35 RNA probe-labeled Sost stain for purposes of detailing theosteoblasts and trabecular bone adjacent to the molar tooth;

FIG. 14F shows a molar tooth bud at a higher magnification stained withS-35 RNA probe-labeled Wise stain for purposes of detailing the dentalfollicle layers;

FIG. 14G shows a bilateral view of two molars on hematoxylin and eosinstaining of a tooth cross-section, an incisor tooth staining patterns,and the morphological features of two incisors, with the nasal crestbetween them, tongue, and hair follicles of the whisker pad;

FIG. 14H shows incisor tooth staining patterns with S-35 RNAprobe-labeled Sost stain for purposes of detailing the osteoblasts oftrabecular bone;

FIG. 14I shows incisor tooth staining patterns with S-35 RNAprobe-labeled Wise stain, prominent Wise staining of incisors, hairfollicles and the whisker pad are also stained with Wise labeled RNAprobes;

FIG. 14J shows X-ray photographs of incisor teeth in the maxilla (upperjaw) regions of the wild type mice, utilizing a 120 strain geneticbackground;

FIG. 14K shows X-ray photographs of incisor teeth in the maxilla (upperjaw) regions of the Wise mutant mice utilizing a 120 strain geneticbackground, the Wise mutant jaw possesses an additional incisor tooth(i′) not present in the wt mouse shown in FIG. 14J, the additional toothmay originate from either an additional tooth bud or, alternatively,from a bifurcation of the original incisor;

FIG. 14L shows the patterning in molar teeth observed in a wt mouseagainst a C57BL6 genetic background;

FIG. 14M shows the patterning in molar teeth observed in a Wise mutantmouse against a C57BL6 genetic background, the additional M1 molar inthe Wise mutant is present compared to the M1, M2, and M3 molars presentin the wt mouse in FIG. 14L;

FIG. 14N shows the patterning in molar teeth observed in a wt mouseagainst a 129 background; and

FIG. 14O shows the patterning in molar teeth observed in a Wise mutantmouse against a 129 background, abnormalities are present compared tothe wt mouse of FIG. 14N.

DETAILED DESCRIPTION

The present invention relates to a family of nucleic acid molecules,which encode polypeptides that bind to LRP and likely regulate the Wntpathway and, resultingly, regulate bone deposition. The polypeptideswill also regulate ocular and tooth development. The present inventionfurther relates to proteins and polypeptides, or amino acid sequences,expressed from the family of nucleic acid molecules, which regulate bonedeposition through LRP interaction. In particular, a nucleic acidmolecule family, which includes the Wise and Sost genes, can be usedwith the present invention, as well as the family of amino acidsequences expressed therefrom. When the above family of amino acidsequences, including Wise and Sost, are allowed to bind to an LRPprotein, bone deposition is regulated. When the family of amino acidsequences are prevented from binding to an LRP protein, deposition ofbone will increase.

Antisense RNAs or oligonucleotides can be used to block translation ofmRNA related to or translated from the above described nucleic acidmolecules—in particular, the LRP binding family of amino acid sequencesand polypeptides can cause increased bone deposition and likely activatethe LRP/Wnt pathway. Similarly, inhibitor peptides and polypeptidesprevent the above family of amino acid sequences from binding to an LRPto thereby increase bone deposition. As such, the present inventionincludes the above listed methods, nucleic acid molecules, amino acidsequence or polypeptide molecules, as well as related compositions andmethods designed to prevent or inhibit binding by the LRP bindingprotein family to LRP. These tools can also be used to effect phenotypicchanges. Specifically, mutants versions of Wise or Sost will causephenotypic changes. Kits are described for detection of the above nativenucleic acid molecules and amino acid sequence molecules. Kits aredescribed for detection of mutant or variant forms of the aforementionednucleic acid molecules, detection of expressed polypeptides or proteins,and measurement of corresponding levels of protein expression.

The novel Wnt inhibitor, Wise, has been isolated in the presentinvention. Wise affects craniofacial anterior-posterior patterning. Thebiochemical function of craniofacial A-P patterning is generallyaddressed in the present invention. Previously, it was shown that whenchick somites were transplanted to more anterior locations, an anteriorshift in Hox gene expression was observed. This shift in expressionresulted in a posteriorization of the more anterior neural tissue. Ascreen for molecules involved in this process lead to the isolation ofWise. Wise is a secreted molecule that, until now, has not been shown toshare much homology to any known molecules. Its gene structure containstwo exons (200 and 400 bp) with a large 2.5 Kb intron (FIG. 10). Thesecond exon encodes a cysteine knot motif, which bears some homology toknown DAN, and CCN family members (FIGS. 9, 10, 11). Wise is mapped toHuman chromosome 7p21.1, which in turn is linked to the HoxA cluster by10.6 Mb (FIG. 9C). The four mammalian Hox clusters are thought to haveevolved from a single cluster, as in Drosophila, therefore otherclusters were searched for a possible Wise family member. Nothing wasfound that linked to the HoxD cluster, however, it was found that bothHoxB and HoxC clusters had an ORF that was examined further. The HoxCcluster ORF, at 4 Mb upstream shares homology to the CCN family. TheHoxB cluster contained an ORF at 5 Mb upstream. The HoxB ORF encodes aknown gene, Sost. Sost was positionally cloned because of a familialmutation affecting bone density. Sost and Wise both share the same genestructure, and produce a secreted protein whose second exon (70%homologous) encodes for a cysteine knot. Unlike the known cysteine knotfrom DAN or CCN family members, Wise and Sost cysteine knots contain 8cysteines instead of 9 like CCN and DAN families. Other molecules,Mucin2 and VWF have cysteine knots containing 10 cysteines, but arearranged in a manner similar to both the CCN and DAN family. DAN and CCNcysteine knots share about 50% homology to those of Wise and Sost. Inaddition to the cysteine knot domain, CCN proteins also encode forInsulin binding, Von Willderbrand, and TSP1 domains. However, the DANfamily appears to only encode for a cysteine knot domain. Other genesthat encode a cysteine knot domain include Slits, VWF, Mucins, and NDP.

A new Wise family member, Sost, has been characterized herein. Both Wiseand Sost are linked to a Hox cluster further supporting Hox clusterduplication hypotheses. Sost functions like Wise to inhibit the Wntpathway, however, unlike Wise, Sost is unable to induce En2 expression.The inability to induce En2 is very similar to other cysteine knotfamily members, like CTGF and Nov.

A family of genes and related proteins or polypeptides was isolated,which likely bind to LRP and likely regulates the LRP/Wnt pathway andcauses regulation of bone deposition. The family of genes includes NDP,Dan, Caronte, PDGF, Wise, Sost, Cereberus, Gremlin, CTGF, Soggy, Dkk1,Cyr61, Dkk2, Dkk3, Dkk4, Nov, Mucin, Slit, OH, WISP, and CCN. Proteinsexpressed therefrom form a related amino acid sequence family. Thesenucleic acid molecules include sequences identified as SEQ ID NOs 1-44,96-103, 105, 108, 110-113, and 126-128, and amino acid sequencesidentified as SEQ ID NOs 45-95, 104-107, 109, 114-125. When the abovegenes of the family are turned off, or mutagenized, the LRP pathwaytypically is not regulated and deposition of bone will increase. Moreparticularly, the gene-encoded proteins do not bind to LRP, resulting inincreased bone deposition. The gene-expressed proteins can be blocked toprevent regulation of the LRP pathway. Thus, the present inventionrelates to nucleic acid molecules and amino acid sequences and othertools and methods used to inhibit, block or deactivate binding of theLRP binding family to LRP. Inhibition of Wnt signaling can occur withresultant blocking or deactivation of the LRP binding family to LRP.

Related to this, it is known that mutant Wise and Sost polypeptidescause phenotypic changes in bone deposition, ocular development andtooth development. Regardless of interaction with LRP it is determinedthat mutants of Sost or Wise, or antibodies which attach to Sost orWise, will cause phenotypic changes.

The above gene family and related proteins can not only be characterizedas binding to or blocking binding to LRP, but as a gene family thatexpresses related proteins that each possess at least one cysteine knot.The cysteine knot is generally formed by 8 cysteine residues, which arereadily conserved. However, other knots may have fewer or more residues.Typically, a guanine is part of the structure and conserved. Guaninewill, along with two other amino acids, separate two cysteines locatedin one arm. For example, the gene family contains the genes Sost, Wise,Dkk1, Dkk2, OH, WISP, and CTGF. These genes include an exon region(e.g., Exon 2), which expresses a protein or amino acid sequencemolecule, which has a cysteine knot and binds to LRP.

Wise genes and polypeptides that have been specifically isolated,including wild types, alleles, mutants, synthetic versions and any otherrelated homologous nucleic acid sequences, are used herewith. Wisecontains two exons, with Exon 2 considered the most important. Exon 2,when expressed, produces a polypeptide that has a cysteine knot.

The present invention includes the LRP binding family of polypeptidemolecules, such as Wise, Sost, Dkk1, Dkk2, and CTGF, that binds to LRP,which will, in turn, likely bind to Wnt. The LRP proteins and relatedgenes will include LRP 1-11, and Arrow. LRPs that have been found to bespecifically related to the present include LRP1, 2, 5, and 6. AvailableLRP nucleic acid sequence, are SEQ ID NOs 29-43, polypeptide SEQ ID Nos67-88.

The present invention also relates to antisense RNA (asRNA)complementary to an mRNA from the LRP binding nucleic acid family, inparticular Wise and Sost, whereby the asRNA will inhibit the members. AnRNA may also be used to induce post-transcriptional gene silencing. ThisRNAi will cause translation of the gene family to cease. Any RNA/DNAthat is complementary to the mRNA related to the discussed gene family,can be used to destroy a family member. Other mutants include pointframe shift, deletion, truncated, base substituted, and less of functionmutations. The loss of function mutations are made with a stop codon.Additionally, a polyclonal or monoclonal anti-peptide antibody to thecysteine knot antigenic region may be used for detection or inhibition.This antibody would inhibit interaction with LRP. The antibody can alsobe directed to the entire Wise or Sost polypeptide. A point mutation maybe made in a nucleic acid sequence member of the gene family, wherebythe expressed protein or polypeptide cannot bind LRP. Alternatively, anantisense oligonucleotide can be used, which will prevent translation ofmRNA and thereby inhibit binding to LRP. An anti-polypeptide antibodycan be used to bind to LRP and prevent binding with a cysteine knotprotein, preferably functioning by a steric hinderance mechanism.

Mutant alleles of the LRP binding gene family can express a protein oramino acid sequence that will not bind LRP and thereby increase bonedeposition. As discussed, expression of such a mutant can betherapeutically desirable, especially when used as a method forproducing stronger bones or increased recovery from bone disease. Thus,the present invention relates to mutants of the listed gene family. Thepresent invention includes administration of such mutant polypeptideproducts that can result in increased bone deposition.

Antibodies, which specifically bind to the above proteins and probes forisolating the proteins or nucleic acid molecules, are further part ofthe present invention. Fab fragments can be derived from the antibodies.Yet another part of the present invention relates to methods forincreasing bone deposition by preventing the protein family from bindingto an LRP and, in turn, likely regulating the Wnt pathway. The inventionincludes methods for blocking expression of the nucleic acid molecules,and methods for preventing the amino acid sequences from binding to Wntor LRP. Kits are also part of the invention which detect mutants andnon-mutants of the nucleic acid molecules, and their expressed aminoacid sequences or polypeptide molecules. As such, the present inventionincludes diagnostic and therapeutic methods and kits for the prediction,assessment, and regulation of bone deposition.

Nucleic acid sequences complementary to the previously listed nucleicacid molecules, preferably the mutants, of the gene family may also beused with the present invention. As expected, such a complementarynucleic acid sequence is one that can be expressed to form a protein oramino acid sequence that binds to LRP and regulates bone deposition. Thecomplementary sequence can also be used to prevent binding of LRP and,thus, increase bone deposition. A complementary nucleic acid sequencefrom a member of the LRP binding gene family can be made to produce anexpressed polypeptide that can impact binding to LRP and ultimatelyregulate bone deposition. Further, degenerate variants of the sequencesmay be used. Also, isolated nucleic acid molecules that encode the LRPbinding family protein or amino acid sequence may be used in the presentinvention.

Nucleic acid molecules homologous to the wild type nucleic acidmolecules, and the mutant nucleic acid molecules, may be used toregulate or cause increased bone deposition. The homologous nucleic acidmolecules are identified using a BLAST (Basic Alignment Search Tool)(NCBI) sequence method. Suitable homology will include those nucleicacid molecules that are 50% homologous to the listed mutant alleles, ornon-mutants. More preferably, the homology will be 60% and, even morepreferred, 75% homologous to the mutant alleles, or non-mutants. Themost preferred homologous nucleic acid molecule will be 90% homologousto the mutant alleles, or non-mutants (i.e. wild type), in particular,Wise, Sost, and mutants thereof. Homologous nucleic acid molecules maybe derived from animals, including, but not limited to, humans,non-human mammals, amphibians, and insects.

Isolated nucleic acid sequences, such as oligonucleotides, can bederived from the nucleic acid molecules, which are the active portionsof the molecules, to bind with LRP, mRNA, or ultimately prevent bindingof the LRP protein. Such oligonucleotides are a part of the presentinvention. The active region, which forms the oligonucleotide molecules,includes the cysteine knot region. More particularly, a region whichexpresses a cysteine knot sequence that binds to LRP can be used.Conversely, oligonucleotides related to the mutant forms of the genescan be used to prevent regulation of bone deposition.

Expression vectors, which regulate bone deposition, can be formed thatcontain the above-discussed nucleic acid molecules, using knownprocedures. A promoter can be operably linked to the isolated nucleicacid molecule to form the expression vector. Any promoter can be usedwhich causes expression of the nucleic acid molecule, and can beswitched on and off. It is further preferred to include a marker withthe vector. Suitable vectors include DNA vectors, plasmid vectors, andshuttle vectors.

Vectors are formed from both the isolated nucleic acid sequences and themutant versions of the isolated nucleic acid sequences. The vectorsinclude expression, cloning, and viral vectors. Other available vectorsinclude fusion vectors, gene therapy vectors, two-hybrid vectors,reverse two-hybrid vectors, sequencing vectors, and cloning vectors.Also, prokaryotic and eukaryotic vectors may be used. Specificprokaryotic vectors that may be used in the present invention includepET, pET28, pcDNA3.1N5-His-TOPO, pCS2+, pcDNA II, pSL301, pSE280,pSE380, pSE420, pTrcHis, pRSET, pGEMEX-1, pGEMEX-2, pTrc99A, pKK223-3,pGEX, pEZZ18, pRIT2T, pMC1871, pKK233-2, pKK38801, and pProEx-HT.Specific eukaryotic vectors that may be used herein include pFastBac,pFastBac HT, pFastBac DUAL, pSFV, pTet-Splice, pEUK-C1, pPUR, pMAM,pMAMneo, pBI101, pBI121, pDR2, pCMVEBNA, YACneo, pSVK3, pSVL, pMSG,pCH110, pKK232-8, p3′SS, pBlueBacIII, pCDM8, pcDNA1, pZeoSV, pcDNA3,pREP4, pCEP4, and pEBVHis. As mentioned, a variety of promoters may beused with the vector, as well as any of a variety of selectable markers.Available markers include antibiotic resistance genes, a tRNA gene,auxotrophic genes, toxic genes, phenotypic markers, colorimetricmarkers, antisence oligonucleotides, restriction endonuclease, enzymecleavage sites, protein binding sites, and immunoglobulin binding sites.Specific selectable markers available include LacZ, neo, Fc, DIG, Myc,and FLAG.

Once the vectors are formed, they can be used to transfect a host cell,whereby a transgenic host cell will be produced that incorporates avector that expresses the selected nucleic acid molecule, which preventsor causes bone deposition through interaction with the LRP. Such bonedeposition may likely involve interaction with the Wnt pathway. Methodsfor transfecting the host cell are well known to those of skill in theart, and comprise culturing the vectors with the host cells.

The host cell can be derived from any of a variety of eukaryotic cellorigins, including animal-, mammalian-, amphibian-, or insect-derivedcells. More preferably, the host cells are derived from non-humanmammals and humans. The preferred host cell is an osteoblast/osteoclast,chrondocytes.

Any of a variety of host cells, including prokaryotic and eukaryoticcells, can be transfected with the vectors previously mentioned.Prokaryotic host cells include Gram-negative and Gram-positivebacteriums may be transfected with any of the variety of the vectorspreviously mentioned. Available bacteriums include Escherichia,Salmonella, Proteus, Clostridium, Klebsiella, Bacillus, Streptomyces,and Pseudomonas. A preferred Gram-negative bacterium is Escherichiacoli. Eukaryotic vectors can be used to transfect eukaryotic host cellsincluding yeast, plant, fish, mammalian, human, mouse, frog, or insectcells. Specific host cells that can be transfected include ES, COS, HEK293, CHO, SaOS, osteosarcomas, KS483, MG-63, primary osteoblasts,osteoclasts, chrondocytes, and human or mammalian bone marrow stroma. Assuch, the present invention includes host cells transfected with any ofthe previously mentioned vectors.

A transgenic animal can be formed using the present invention. Inparticular, transgenic non-human animals can be formed by insertion ofthe wild type or mutant nucleic acid molecules into cells of a hostanimal. The insertion of nucleic acid molecules into host animal cellscan occur by a variety of methods including but not limited totransfection, particle bombardment, electroporation, and microinjection.Insertions can be made into germ line, embryonic, or mature adult hostanimal cells.

The proteins or amino acid sequences expressed by the nucleic acidmolecules, related mutants, and the listed nucleic acid molecules canactivate LRP/Wnt and can be isolated and purified. Additionally, themutants, asRNA molecules, as oligonucleotides, and anti-peptideantibodies can be developed and used to prevent binding to LRP orbinding of Wise or Sost. The proteins or amino acid sequences from boththe non-mutant and mutant nucleic acid molecules can also be isolatedand purified. Such isolation and purification include known proceduresand methods, including affinity chromotography or purification, as wellas other methods. The isolated proteins include those listed herein.Additionally, suitable proteins or amino acid sequences include thosethat bind to LRP and Wnt, and prevent or cause activation, dependentupon the desired outcome.

Proteins, which are 90% homologous with the polypeptides listed in SEQIDs are also included. As would be expected, polypeptides or proteinsthat are 50% homologous to the polypeptides may also be used, withproteins 60% homologous more preferred. A polypeptide that is 75%homologous to SEQ ID NOs 45-95, 104-107, 109, and 114-125 is even morepreferred. As such, any of a variety of polypeptides may be used, aslong as they are expressed by an LRP binding family member, Sost Wise,or homologous nucleic acid molecule, and prevent influence Wnt, Bonedeposition, tooth development or ocular development. More preferably,mutants will be used. Resultingly, the proteins will cause increases ofbone deposition to occur. Non-mutant, homologous amino acid sequencesmay be used. The extent of homology will be identical to that previouslydescribed above. Thus, sequences that are 50% homologous to the proteinsor amino acid sequences may also be formed. More preferably, thesequences will be 75% homologous, and even more preferably, 90%homologous to the proteins.

Probes, which can be used to isolate, identify, and characterize theabove proteins and/or genes, can be formed from such proteins or genes.The probes include cDNA, mRNA, and monoclonal and polyclonal antibodies.All the probes are formed using known procedures. Probes, which are 50%homologous to the proteins or amino acid sequence, may also be formed.More preferably, the probes will be 75% and, even more preferably, 90%homologous to the above proteins. The formula used to determine thehomology of the probes is a BLAST sequence.

Antibodies, which specifically bind to the above-listed proteins, arepart of the present invention. Additionally, hybridomas that producesuch antibodies are used herewith. In addition to protein probes, cDNAprobes may be formed, which are comprised of isolated nucleic acidmolecules previously discussed. As such, any antibody that bindsspecifically to a Wnt binding family member, may be used. Antibodiesthat selectively bind to an epitope in the receptor-binding domain ofthe LRP/Wnt binding mutant protein may also be used. A non-mutant orwild type epitope may also be used.

A kit for detecting a LRP binding gene, or related nucleic acidmolecule, can be formed. The kit will preferably have a container and anucleic acid molecule, which includes any of the mentioned sequences.

A kit for detecting a LRP binding protein or amino acid molecule canalso be formed. The kit will preferably have a container and a nucleicacid molecule, which includes any of the mentioned sequences.

The family of genes and proteins can be used as tools to develop asRNAsand polypeptides, which regulate LRP/Wnt.

Neural patterning in embryogenesis involves signaling between the neuralplate and surrounding tissues. To investigate this process, a functionalscreen was performed using a cDNA library derived from chick tissuessurrounding the neural tube. Activities that alter anteroposterior (A-P)character of neuralized Xenopus animal caps were assayed for, and anovel gene was identified, Wise, which was expressed in surfaceectoderm. Wise encodes a secreted protein capable of inducing posteriorneural markers. Importantly, the phenotypes arising from ectopicexpression of Wise resemble those affected when Wnt signaling isaltered. Induction of posterior markers by Wise likely requirescomponents of the canonical Wnt pathway, showing that it activates theWnt signaling cascade. In contrast, in other assays, such as secondaryaxis induction, Wise inhibits Wnt signaling. Wise protein interacts withLRP receptors, but not with Wnt, demonstrating that Wise is a novelligand for LRP, which either activates or inhibits the signalingpathway. Hence, Wise differentially influences the Wnt signaling cascadein a context-dependent manner. These activities provide a novelmechanism that integrates and modulates the balance of Wnt signaling.

The following are definitions for terms used herein.

An animal cap is a pigmented animal hemisphere of the amphibianblastula. The vegetal becomes endoderm and part of the animal polebecomes ectoderm. In most animal oocytes the nucleus is not centrallyplaced, and its position can be used to define two poles. That nearestto the nucleus is the animal pole, and the other is the vegetal pole,with the animal-vegetal axis between the poles passing through thenucleus. During meiosis of the oocyte, the polar bodies are expelled atthe animal pole. In many eggs, there is also a graded distribution ofsubstances along this axis, with pigment granules often concentrated inthe animal half and yolk region, when present, largely situated in thevegetal half.

The anterior-posterior axis is the body axis extending from the anteriorto the posterior pole of a bilaterally symmetric embryo (or animal).

Blastomere is one of the cells produced as the result of cell divisionand cleavage, in the fertilized egg.

Blastula is the stage of embryonic development of animals near the endof cleavage but before gastrulation. In animals where cleavage or celldivision involves the whole egg, the blastula usually consists of ahollow ball of cells.

Bone is continually deposited by osteoblasts. Normally, bone depositionand absorption are equal.

DNA cassette is a deoxyribonucleic acid (DNA) sequence that can beinserted into a cell's DNA sequence. The cell in which the DNA cassetteis inserted can be a prokaryotic or eukaryotic cell. The prokaryoticcell may be a bacterial cell. The DNA cassette may include one or moremarkers, such as Neo and/or LacZ. The cassette may contain stop codons.In particular, a Neo-LacZ cassette is a DNA cassette that can beinserted into a cell's DNA sequence located in a bacterial artificialchromosome (BAC). Such DNA cassettes can be used in homologousrecombination to insert specific DNA sequences into targeted areas inknown genes.

The ectoderm is the germ layer that gives rise to the epidermis andnervous tissue.

The endoderm is the germ layer that gives rise to the respiratoryorgans, gut, and the gut accessory glands.

Gastrula is the stage of embryonic developments in animals whengastrulation occurs, and follows the blastula stage.

Gastrulation is the process by which cells of the blastoderm aretranslocated to new positions in the embryo, producing the three primarygerm layers.

The germ layer is defined as the main divisions of tissue types inmulticellular organisms. Diploblastic organisms (e.g., coelenterates)have two layers, ectoderm and endoderm; triploblastic organisms (i.e.,all higher animal groups) have mesoderm between these two layers. Germlayers become distinguishable during late blastula/early gastrula stagesof embryogenesis, and each gives rise to a characteristic set oftissues, the ectoderm to external epithelia and to the nervous system,for example, although some tissues contain elements derived from twolayers.

Mesoderm is defined as the middle of the three germ layers; which givesrise to the musculo-skeletal, vascular, and urinogenital systems, toconnective tissue (including that of dermis) and contributes to somegland formation.

Neural plate is defined as a region of embryonic ectodermal cells,called neuroectoderm, that lie directly above the notochord. Duringneuralation, the neuroectoderm changes shape, so as to produce aninfolding of the neural plate (i.e., the neural fold) that then seals toform the neural tube.

The neural tube is the progenitor of the central nervous system.

Somites are defined as the blocks of tissue in the trunk derived fromthe originally unsegmented paraxial mesoderm.

Small molecules are defined as regulatory polypeptide or nucleic acidmolecules that cause detectable changes in protein-protein interactionsystems that may also affect one or more phenotypic changes. Interactionsystems include, but are not limited to, Wise and Sost proteininteraction with LRPs, the Wnt pathway, Engrailed, and Frizzled. Thesesmall molecules may operatively function by structural similarity to andcompetitive inhibition with native molecules in vitro or in vivo.Phenotypic changes may include observed changes in such parameters asbone deposition or bone mineral density, tooth development, and oculardevelopment. Small regulatory polypeptide molecules include, but are notlimited to, antibody fragments such as Fab, F(ab)₂, Fv, and antibodycombining regions that bind with either Wise, Sost, or LRP; andshortened Wise, Sost or LRP polypeptide sequences. Small regulatorynucleic acid molecules include, but are not limited to, antisense RNAsequences that interfere with Wise, Sost, or LRP function; and truncatedWise, Sost or LRP nucleic acid sequences that encode shortenedpolypeptides that interfere with Wise; Sost or LRP function. Anantisense Wise RNA is complementary to Wise sense RNA and operativelybinds to it in a cell to prevent translation of native protein. Atruncated Wise nucleic acid sequence encodes a shortened Wisepolypeptide that can potentially competitively bind to LRP to preventnative Wise protein binding.

The vegetal pole is the surface of the egg opposite to the animal pole.Often the cytoplasm in this region is incorporated into future endodermcells.

A vector is a self-replication DNA molecule that transfers a DNA segmentto a host cell.

A host organism is an organism that receives a foreign biologicalmolecule, including an antibody or genetic construct, such as a vectorcontaining a gene.

Chimera is an individual composed of a mixture of genetically differentcells. The genetically different cells of chimeras are required to bederived from genetically different zygotes.

Mutant is an organism bearing a mutant gene that expresses itself in thephenotype of the organism. Mutants include both changes to a nucleicacid sequence, as well as elimination of a sequence. In additionpolypeptides can be expressed from the mutants.

Plasmids are double-stranded, closed DNA molecules ranging in size from1 to 200 kilo-bases. Plasmids are vectors for transfecting a host with anucleic acid molecule.

An amino acid (aminocarboxylic acid) is a component of proteins andpeptides. Joining together of amino acids forms polypetides. Polymerscontaining 50 or more amino acids are called proteins. All amino acidscontain a central carbon atom to which an amino group, a carboxyl group,and a hydrogen atom are attached. Polypeptides can be referred to when aprotein is less than 500 amino acids.

A nucleic acid is a nucleotide polymer better known as one of themonomeric units from which DNA or RNA polymers are constructed, itconsists of a purine or pyrimidine base, a pentose, and a phosphoricacid group.

A gene is a hereditary unit that has one or more specific effects uponthe phenotype of the organism that can mutate to various allelic forms.

A polypeptide is a polymer made up of less than 50 amino acids.

Knockout is an informal term coined for the generation of a mutantorganism (generally a mouse) containing a null allele of a gene understudy. Usually the animal is genetically engineered with specifiedwild-type alleles replaced with mutated ones.

Allele is a shorthand form for allelomorph, which is one of a series ofpossible alternative forms for a given gene differing in the DNAsequence and affecting the functioning of a single product.

Wild type is the most frequently observed phenotype, or the onearbitrarily designated as “normal”. Often symbolized by “+” or “wt.”

Finally, the phenotypes observed in Wise mutants are similar to that ofSost mutants. Some phenotypes examined in the Wise mutant may explainSost phenotypes, i.e. loss of retinal nerve fibers may be reason foroptic nerve atrophy. Interestingly, it has been demonstrated that Wiseinhibits the Wnt pathway by binding to an area encompassing the firsttwo YWTD propeller domains of LRP. In humans the autosomal recessivedisorder OPPG has been mapped to the area upstream of the first YWTDpropeller domain of LRP5. Also, LRP5 is found to be expressed inosteoblasts and in retinal cells of Xenopus embryos. The same expressionpattern was found for humans. It has been demonstrated that the loss ofLRP5 function leads to very low peak bone mass and visual loss. Thus,early during bone development, Wise may be acting to inhibit Wntsthrough LRP5; and later, the inhibition of Wnts may be the function ofSost.

EXAMPLES Example 1

Functional screens in Xenopus were performed with the aim of identifyingfactors derived from tissues surrounding the neural tube that alter A-Ppatterning in Noggin-treated animal caps. Two clones were isolated, oneencoded a truncated β-catenin and the other a novel secreted protein,which was named Wise. Isolation of the two clones is described below.

FIG. 1A provides an overview of how factors which impacted patterningwere determined. Chick embryo somites, which are capable of transformingpre-otic rhombomeres into a more posterior neural tissue were collectedtogether with overlying ectoderm and underlying endoderm. mRNA wascollected from the tissue, which was then used to make a cDNA library.This provided a source of putative posteriorizing factors.

The cDNA library was made from stage 8-13, (Hamburger and Hamilton,1951) chick embryos using tissues surrounding the neural tube (FIG. 1A)from axial levels capable of inducing Hoxb9 expression in graftingexperiments (Itasaki et al., 1996). The library contained 250,000un-amplified clones, and 50,000 of these were divided into 100 pools(500 clones per pool). For initial screening, 10 pools were mixed toprepare a single large DNA pool (5,000 clones) used to synthesize cappedRNA. Size-selected (>1 kb) cDNAs were directionally inserted into amodified 64T vector (Tada et al., 1998).

Xenopus eggs were obtained, fertilized, cultured, and injected with thesynethized RNA, as previously described (Jones and Smith, 1999). In thefirst round of screening, 250 picogram (pg) of Noggin RNA and 12nanograms (ng) of library RNA were injected into each blastomere of2-cell state Xenopus embryo. To examine embryo phenotypes, RNA wasinjected into specific blastomeres, together with lacZ or FIDx(Molecular Probes) as a lineage tracer. Markers were assayed with insitu hybridization.

Following co-injection of Noggin RNA with pools of RNA from the cDNAlibrary, the induction of posterior markers was monitored in animal capsby assaying for expression of En2, Krox20, and Hoxb9, which mark themidbrain, hindbrain, and spinal cord, respectively (FIGS. 1B and 1C).Myosin was also used as a marker for mesoderm induction, which allowedfocus on pools that influence neural patterning in the absence ofmesoderm.

Explants (excised tissue) were processed for RT-PCR to detectregion-specific neural markers. The primers for Ef1α, NCAM, Otx2, En2,Krox20, Hoxb9, Myosin light chain and Muscle actin were used.

It was observed in pool 5, that En2 was induced in the absence ofmesoderm (FIG. 1B). Successive rounds of sub-division and sib selectionidentified the clones responsible for this activity. From this pool, twodistinct clones were isolated. One clone encoded an amino-terminallytruncated form of β-catenin, a cytoskeletal component, and anintracellular target of the Wnt pathway. This result was consistent withdata demonstrating that β-catenin has an ability to induce posteriorneural markers in animal caps when co-injected with Noggin. TheN-terminal truncation in the clone removed the first 87 amino acids,which included the sites for phosphorylation by GSK3β, which accelerateddegradation of β-catenin protein. Therefore, the clone encoded a stableform of β-catenin able to stimulate Wnt signaling.

The second clone proved to encode a novel protein. Based on itscharacterization and relationship to Wnt signaling detailed in thestudy, the clone's gene was designated Wise (Wnt, inhibitor/activator insurface ectoderm). In the animal cap assays, injection of Wise RNA,together with Noggin, demonstrated that increasing concentrations ofWise progressively induced more posterior markers (En2 and Krox20) inthe absence of mesoderm (FIG. 1C). Noggin equal to 500 pg and Wise equalto 150, 300, 600 and 1200 pg were injected. Wise alone exhibited noneural-inducing activity (no NCAM induction) and no ability to inducemesoderm, as confirmed using Myosin (FIG. 1C), Brachyury, Wnt8, andXhox3 as markers. It was observed that increasing amounts of Wise RNA(150, 300, 600, and 1200 pg) progressively induced more posterior neuralmarkers in the presence of Noggin. Wise DNA and RNA were obtained usingstandard molecular biology methods. Sambrook et al., Molecular Cloning:a Laboratory Manual, 3^(rd) ed., Cold Spring Harbor, N.Y., Cold SpringHarbor Laboratory Press (2001).

For explant recombination assays, 500 pg of Noggin was injected into oneset of embryos and 1 ng of Wise injected into a separate set. Forlineage tracing, either FIDx was injected, along with Noggin RNA, or 100pg of lacZ RNA was co-injected with Wise. Caps were cut at stage 8,combined and cultured for assay at stage 25.

Example 2

To isolate a frog clone, Xenopus stage 25 embryos were collected and acDNA library was formed. This was used as a template for RT-PCR. Usingdegenerate primers, designed on the basis of conserved regions betweenchick and mouse Wise, ˜500 bp fragments were subcloned intopBluescriptIIKS (Stratagene) and sequenced. The degenerate primers usedwere upstream, SEQ ID NO 129: 5′-GCTTT(T/T)AA(A/G)AA(C/T)GATGCCAC-3; anddownstream, SEQ ID NO 130: 5′-GTGAC(T/C)AC(T/G/A)GT(T/G)ATTTTGTA-3′. Twodifferent clones in the frog were identified (XWise-A and XWise-B)presumably resulting from the pseudotetraploid Xenopus genome. For eachclone; 5′ and 3′ flanking sequences were identified by PCR using aXenopus stage 35 cDNA library. Standard PCR methods are described inU.S. Pat. No. 4,683,195; U.S. Pat. No. 4,683,202; Saiki et al., Science230:1350-1354 (1985); Innis et al., PCR Protocols: A Guide to Methodsand Applications, Academic Press, Inc., San Diego, Calif. (1990).

The predicted amino acid sequence of XWise-A was used for comparisonwith other species which are listed in FIG. 2A, which shows Wise as aconserved secreted protein. Various EST databases were searched, withthe predicted amino acid sequences then aligned in FIG. 2A. Thepredicted amino acid sequence of Zebrafish, Xenopus, chick, mouse, andhuman Wise proteins were compared.

The predicted Wise protein, SEQ ID NO 45, consists of 206 amino acidsand contains cysteine knot-like domains. These cysteine knot domains arefound in a number of growth factors, as well as in Slit, Mucin, and CCN(Cef10/Cyr61, CTGF and Nov) family members (Bork, 1993). Among these,the C-terminal domain of the CCN family members showed the highesthomology to Wise, but other motifs conserved within the CCN family wereabsent in Wise (FIG. 2B). Hence, Wise is related to, but not a memberof, the CCN family. A homology search revealed that Wise showed thehighest amino acid identity (38%) to Sclerostin (Sost), identified bypositional cloning of the gene mutated in sclerosteosis (Brunkow et al.,2001).

Wise was further analyzed, as shown in FIG. 2B. The shaded boxes in FIG.2B indicate three blocks (Δ1, Δ2, Δ3) deleted separately for functionalanalysis. This was done to investigate if the conserved regions wererequired for functional activity of Wise, three separate deletions weregenerated, and their ability to induce En2 expression in Noggin-injectedanimal caps was tested. The variant that deleted 19 amino acids outsideof the CT domain (Δ1) retained the ability to induce En2. In contrast,two deletions corresponding to different parts of the Slit homologydomain (Δ2 and Δ3) abolished the ability of Wise to induce En2,demonstrating that these regions were necessary for Wise function.

Example 3

A signal sequence motif is present at the N-terminus of Wise, and itssecretion was confirmed by Western blotting following expression of anHA-tagged version of the protein in Xenopus oocytes (FIG. 2C) and COScells. More particularly, Wise was injected in an amount equal to 30ng/embryo. Western blot analysis detected HA-tagged Wise proteinsecreted into the medium following RNA injection into oocytes. FIG. 2C,related to the control of uninjected oocytes. Secretion of Wise wasconfirmed by expression of an HA-tagged version of the protein inXenopus oocytes and COS cells. The protein was detected in both cellextracts and the culture medium (FIG. 2C). It was observed that Wiseencoded a signal sequence motif at its N-terminus, suggesting that theprotein is secreted.

Further, the ability of Wise to posteriorize neural tissue in a cellnon-autonomous manner was tested by using a tissue recombination assayin which a Wise-expressing animal cap was combined with anoggin-expressing animal cap. It was found that both En2 and Krox20 wereinduced in discrete domains in the Noggin caps (FIGS. 2D and 2E). Nogginwas injected in an amount equal to 500 pg and Wise equal to 600 pg.Hence, it was determined Wise has the ability to induce posteriormarkers at a distance.

Subsequently, the ability of Wise to posteriorize tissues in a cellnon-autonomous manner was tested. Recombination betweenNoggin-expressing and Wise-expressing animal caps were assayed forexpression of Krox20 or En2, FIGS. 2D and 2E respectively. Wise induceda ring of En2 (en) expression or patches of Krox20 staining in anon-cell autonomous manner in the Noggin cap. In FIG. 2D, the Noggininjected cap was marked with FIDx, and in 2E, the Wise cap was markedwith lacZ as lineage tracers. Using a tissue recombination assay inwhich a Wise-expressing animal cap was recombined with a Nogginexpressing animal cap, it was found that both En2 and Krox20 wereinduced in the Noggin caps (FIGS. 2D and 2E). As such, it was determinedthat Wise has the ability to induce posterior markers at a distancethrough the induction of Wnt.

Example 4

The following Example analyzes the expression of Wise in chick andXenopus embryos. Whole mount in situ hybridization analysis and sectionsin stage 9-12 chick embryos revealed that Wise was expressed in adynamic manner in the surface ectoderm (FIGS. 3A-3D). Expression wasdetectable first at stage 9. Expression was localized in the posteriorsurface ectoderm overlying the presomitic mesoderm, wherein somites wereformed by stage 10-11 (FIGS. 3A, 3B, and 3D). FIGS. 3A-3D show in situhybridization of chick embryos. Wise was expressed in the surfaceectoderm from the level of presomitic mesoderm to the posterior end atstage 10, FIG. 3A. Higher transcript levels are detected at stage 11,FIG. 3B, which refine to a small posterior domain by stage 12, FIG. 3C.This is shown by the red stain in the FIG. 6. A section shown in FIG.3D, in the vicinity of Hensen's node, showed Wise transcripts confinedto the surface ectoderm (se). This is shown by the arrow. Expressiondecreased rapidly during stages 12-13, and resolved into a smallposterior domain (FIG. 3D). This expression profile suggested that theoriginal Wise cDNA was derived from the ectodermal part of the tissueused to make the library (FIG. 1A).

In an RNase protection assay, Xenopus Wise expression was weaklydetected initially at gastrula stages (stage 10), and expressionpersisted into tadpole stages (FIG. 3E). FIG. 3E shows an RNaseprotection assay of Xenopus embryos with stages noted above each lane.Wise is first detected at an early gastrula stage, persisting intotadpole stages. ODC was a loading control. In later stage chick embryos,Wise was expressed in branchial arches and other specialized tissues,including feather buds. A similar pattern was observed in Xenopusembryos. Wise was expressed in the surface ectoderm, but had a broaderdomain along the A-P axis, in comparison to chick (FIG. 3F). FIGS. 3Fand 3G show the whole mount in situ hybridization to Xenopus embryos. Atstage 15 (FIG. 3F), Wise is expressed in the surface ectoderm at allanterior-posterior levels. The expression is strongest at the edge ofthe neural tube. At tadpole stages (FIG. 3G, stage 40), expression waslocalized in epibranchial placodes, lateral lines, and along the dorsalfin.

This data showed that Wise caused posterior development. It also showedthe stages of development when Wise had the strongest effect.

Example 5

The present Example relates to changes observed in neuronal markersafter blastomer injections of Wise RNA and Wise antisense morpholinooligos (FIG. 4).

Morpholino antisense oligos were designed against the beginning of thecoding region of Xenopus Wise-A and B. The sequences were: A (SEQ ID NO131), 5′-AGCACTGGAGCCTTGAGACAACCAT-3′; B (SEQ ID NO 132),5′-AGCAGTGAAGCCTTGAGACAACCAT-3′. A 1:1 mixture of these oligos wasdiluted in PIPES (5 mM) buffered water and used for injection. In vitrotranslation of Wise RNA was inhibited at oligo concentrations of between1-10 μM, which is equivalent to injecting 6-60 ng into one Xenopusembryo (1.2 mm diameter). For whole embryos, 30-60 ng of morpholino wasinjected, and for blastomeres (animal-dorsal or animal-ventralblastomere to target the surface extoderm) 13-33 ng was injected.

FIG. 4 shows changes in neuronal markers after blastomere injection ofWise RNA and Wise antisense morpholino oligos (FIGS. 4A-L). In situhybridization with neural markers in stage 16-21 Xenopus embryosfollowing single blastomere injections of Wise RNA (FIGS. 4B, 4E, 4H,and 4K) at the 8-cell stage and antisense morpholino oligos (FIGS. 4C,4F, 4I, and 4L) at the 4-cell stage are shown. The left panels (FIGS.4A, 4D, 4G, and 4J) indicate control embryos. In most embryos, lacZ(blue staining) was co-injected as a lineage tracer. Injected sides wereto the left. Probes were Sox3 (FIGS. 4A-4C), En2 (FIGS. 4D-4F), Krox20(FIGS. 4G-4I), and Slug (FIGS. 4J-4L). In Wise RNA injected embryos, theneural markers were generally displaced posteriorly. Ectopic inductionof Krox20 and Slug can be seen in the forebrain region (FIGS. 4H and4K). In embryos injected with antisense morpholino oligos, these markerswere unchanged.

FIGS. 4M and 4N show the transverse sections at stage 16 afterblastomere injection of either Wise RNA (FIG. 4M) or Wise antisensemorpholino oligo (FIG. 4N). In FIG. 4M, the neural plate on the injectedside was greatly expanded, which is revealed by Sox3 staining (darkblue, *). Conversely, in the morpholino oligo-injected embryo (FIG. 4N),the surface ectoderm is thicker on the injected side (left, *) incomparison to the right control side.

To further evaluate the effects of Wise on development of the neuraltube, RNA or DNA was injected into specific blastomeres at 4-16 cellstages. When Wise RNA injections were targeted to presumptive neuralregions, expression of pan-neural markers (Sox3, NCAM) confirmed anexpansion of the neural plate (FIGS. 4B and 4M). A-P specific markers(En2, Krox20, and Slug) were generally displaced laterally andposteriorly and were frequently expanded (FIGS. 4E, 4H, and 4K).

Identical results were obtained using DNA constructs for injection,where Wise expression commenced at mid-blastula stages under the controlof a cytoskeletal actin promoter. Together, these changes in morphologyand neural patterning demonstrated that ectopic expression of Wisedisturbed extension and closure of the developing neural tube.

The disruption of neural tube morphogenesis made it difficult to assayfor posteriorizing influences in whole embryos. However, when Wiseinjected cells were targeted to the forebrain region, ectopic expressionof Slug and Krox20 was observed (FIGS. 4H and 4K). This indicated thatanterior forebrain cells acquired a more posterior character in responseto Wise.

Localized injection of the morpholino oligo resulted in embryosdeveloping with thickened ectoderm, which contrasted with Wise RNAinjections where embryos developed with a thickened neural plate (FIGS.4M and 4N). Neural markers, such as Sox3, En2, Krox20, and Slug, werenot obviously affected at early neural stages (FIGS. 4C, 4F, 4I, and4L). This verifies that Wise and Wise mutants influence A-P patterning.

Example 6

Like Example 5, Wise RNA and morpholinos were injected into embryos.Injection of Wise RNA and morpholino oligos were observed to impactneural markers. Anterior defects after blastomere injection of Wise RNAor morpholino oligo were observed. Defects in anterior patterning,including a failure in eye formation, were observed at tailbud stages(FIG. 5H). Furthermore, expression of the cement gland marker XCG wasspecifically down-regulated in cells expressing Wise (FIGS. 5B and 5E).Conversely, when Wise injected cells were distributed more ventrally,the ectopic induction of the cement gland and XCG expression wasobserved (FIG. 5B). Therefore, ectopic expression of Wise alteredaspects of A-P patterning in embryos, as well as animal caps.

FIGS. 5A-5L shows in situ hybridization with the cement gland marker XCGat stage 16-20 (FIGS. 5A-5C) and morphological phenotypes of cementgland at stage 26-40 (FIGS. 5D-5F). Hybridization with the eye at stage35-36 is shown at FIGS. 5G-5I. The controls are shown in FIGS. 5A, 5D,and 5G. Blue staining shows co-injected lacZ lineage tracer.Over-expression of Wise resulted in formation of larger cement glands(FIG. 5C). Eye formation is consistently blocked by injection of bothWise RNA (FIG. 5H) and the morpholino oligo (FIG. 5I).

To analyze the endogenous role of Wise in embryogenesis, the Xenopuscognate was isolated and used to design morpholino antisenseoligonucleotides, which would specifically interfere with translation ofWise RNA. In vitro translation of Wise was blocked by the morpholinooligo, whereas a control oligo had no effect (FIG. 5J). FIG. 5J shows invitro translation of Wise in the presence of the Wise morpholinoantisense oligo. Lane 1 shows translation of Wise protein withoutmorpholino oligo. Lanes 2-7 show translation in the presence of the Wisemorpholino oligo at concentrations of 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM,10 μM, respectively. Lane 8 shows translation in the presence of controlmorpholino oligo at the concentration of 10 μM. Wise translation ispartially blocked at concentration of 1 μM, and completely blocked at 10μM.

When the morpholino oligo was injected into the whole embryo at the 1cell stage, embryos developed cyclopic eyes (FIGS. 5L-5N), and the trunkand tail were shortened in most cases (FIGS. 5F and 5L). At laterstages, morpholino-injected embryos showed defects in eye formation(FIG. 5I), which were rescued by co-injection of Wise RNA (FIG. 5K).FIG. 5K shows the rescue of the eye defect resulting from injection ofthe morpholino oligos by co-injection of Wise RNA.

FIGS. 5L-5N are the phenotypes of embryos following injection of Wisemorpholino oligos throughout the whole embryo. FIG. 5L shows the rangeof cyclopic eye and short trunk phenotypes induced by the oligos incomparison to the control embryo (left). Section of control (FIG. 5M)and morpholino-injected (FIG. 5N) embryos at the level of eye are shown.In the Wise morpholino-injected embryos, eyes are positioned very closeto the neural tube.

These results suggest that the endogenous role of Wise is to mediateelongation of the trunk, morphogenesis of the ectoderm/neuroectoderm,and formation of the eye. The fact that both ectopic expression of Wise,and inhibiting its function by injection of the antisense morpholinooligo resulted in similar defects in eye formation, suggests that thisprocess requires a precise level of signaling, mediated by Wise.

Example 7

The present Example relates to the immunoprecipitation procedurespreviously discussed. To test protein secretion, RNA encoding the HAtagged version of Wise was synthesized and injected into Xenopusoocytes. This HA tagged Wise construct was confirmed to be functional bytesting its ability to induce En2 in Noggin-injected animal caps.Fifteen oocytes were incubated in 96-well dish with 150 μl of OR2medium+0.01% BSA for 2 days. Oocytes and the conditioned medium werecollected separately and used for Western blotting with an anti-HAantibody (Boehringer). This construct was also transfected into COScells and assayed for secretion by Western blotting.

For protein interaction studies, COS cells were transfected with DNAconstructs encoding tagged versions of the proteins. Cells wereharvested and proteins were extracted in 150 mM NaCl, 1% NP40, 0.5%Sodium Deoxycholate, 0.1% SDS, 50 mM Tris-HCl (pH8), a cocktail ofprotease inhibitors (Complete, Boehringer), and 1 mM AEBSF at 4° C.Small aliquots were kept as cell extracts for checking expression ofeach protein. Primary antibodies against the epitope and ProteinA-coupled beads were added to the extracts, incubated for 2 hours, andcollected by centrifugation. Following several rounds of washing,pellets were resuspended in loading buffer in the presence of SDS andsubjected to electrophoresis and Western blotting. The proteins weredetected by using the epitope-specific antibodies and appropriatesecondary antibodies conjugated to alkaline phosphatase.

Example 8

The ability of Wise to interact with the Wnt pathway, and the fact thatit is normally expressed in a transient manner in the non-neural surfaceectoderm, suggest that it might have a role in modulating Wnt signalingin this tissue (FIG. 3). A balance between Wnt and BMP signaling in thesurface ectoderm and dorsal neural tube is important in modulatingdorsal fates and the generation of neural crest cells. Furthermore, Wntsin the surface ectoderm influence patterning of the underlying somitesand their derivatives. The distribution and timing of Wise expression inthe surface ectoderm, together with the result of morpholinoexperiments, suggest that it promotes precise levels of Wnt signaling tocontrol some of these interactions.

FIGS. 6A-6C show RT-PCR of Noggin treated animal caps assayed for En2(en) induction. NCAM is used as a pan neural marker and Ef1a is aloading control. FIG. 6A shows the induction of En2 by Wnt8 or Wise RNAis blocked by dominant-negative (dn) Frizzled 8 (ΔFz8). Noggin was addedin an amount equal to (500 pg); Wnt8 was (50 pg); Wise was (1.2 ng); andΔFz8 (2 ng). In FIG. 6B, the following constituents were added: Noggin(500 pg); Wise (600 pg); ΔWnt8 (200 pg); ΔDsh(dd1) (1.2 ng); GSK3 (500pg); and LEFΔN (200 pg). FIG. 6B shows the induction of En2 is blockedby dn-Wnt8 (ΔWnt8), dn-dishevelled (ΔDsh(dd1)), GSK3 and dn-Lef1(LEFΔN). FIG. 6C shows the induction of En2 requires signalingcomponents of the canonical Wnt pathway but not the planar cell polarity(PCP) pathway. Wise-mediated En2 induction was abolished by ΔDsh(dd1), adominant negative form of Dishevelled for both pathways, and ΔDsh(DIX),which blocks the canonical pathway. ΔDsh(DEP) blocks the PCP pathway buthas no effect on Wise induction of En2. ΔDsh(ΔN) specifically activatesthe PCP pathway but fails to induce En2 in the absence of Wise, althoughfull length d

Dishevelled (Dsh) is able to do so. In FIG. 6C, the followingconstituents were added: Noggin (500 pg); Wise (1.2 ng); Dsh (1 ng); andΔDsh(d1), ΔDsh(DIX) and ΔDsh(DEP) (2.0 ng). FIGS. 6D-6G: TCF3 (300 pg);Wnt8 (25 pg); Wise (300 pg); and β-catenin (100 pg) were added in thelisted amounts.

FIGS. 6D-6G show staining for sub-cellular localization of endogenousβ-catenin detected immunocytochemically in Xenopus animal caps followingRNA injection of: D, TCF3; E, Wnt8+TCF3; F, Wise+TCF3; and G,β-catenin+TCF3. Wnt8 (E) and Wise (F) promoted accumulation of nuclearβ-catenin.

Wise activated the Wnt signaling pathway in animal caps. Since Wnts andWise both induced En2 expression in Noggin-injected animal caps, whetherWise required Wnt signaling for its activity was investigated. To test,Wise RNA was co-injected with either wild-type GSK3β or dominantnegative (dn) versions of the canonical Wnt pathway components, Wnt8,Frizzled, Dishevelled or Lef1. All of these Wnt blocking reagentseliminated the ability of Wise to induce En2 in neuralized animal caps(FIGS. 6A and 6B). The finding that dn-Wnt8 and dn-Frizzled8 blockedWise activity implied that it may use the same receptor(s) as Wnt. Withrespect to the intracellular components, Dishevelled (Dsh) is animportant branch point in Wnt signaling that separates the canonicalnuclear pathway from a planar cell polarity (PCP) pathway. Differenttruncated dishevelled constructs were used to examine the roles of thedifferent pathways in En2 induction. Both ΔDsh (dd1), which lacks a partof the PDZ domain necessary for both the canonical pathway and the PCPpathway, and ΔDsh (DIX), which is a specific dominant negative form forthe canonical pathway, abolished En2 induction by Wise (FIGS. 6B and6C). In contrast, both ΔDsh(DEP), which specifically blocks the PCPpathway, and ΔDsh (ΔN) which constitutively activates the PCP pathway,had no effect on En2 induction (FIG. 6C). These results suggested thatthe domains of Dsh, critical for the canonical Wnt signaling pathway,are essential for Wise function.

Example 9

This Example demonstrates that expression of Wise interferes with Wntsignaling. Although induction of En2 can be explained in terms ofactivation of Wnt signaling, the effects of injected Wise RNA on cementgland formation (FIG. 5B) resemble those seen when the Wnt pathway isinhibited. Therefore, it is possible that Wise also inhibits Wntsignaling. As such, Wise's ability to antagonize Wnt8 activity in axialinduction was examined.

In particular, FIGS. 7A-7C show the secondary axes induced by Wnt8 areblocked by Wise. Injection of Wnt8 RNA into a ventral vegetal blastomereof 4-8-cell stage embryos induced complete secondary axis formation(FIG. 7A). Co-injecting Wise blocked formation of Wnt8-induced secondaryaxis (FIG. 7B), similar to co-injection of a dominant negativeDishevelled, ΔDsh(DIX) (FIG. 7C).

FIGS. 7A-7C show Wnt8 (5 pg); Wise (200 pg); and ΔDsh(DIX) (1 ng) thatwere added in the listed amounts. In FIG. 7D Wise (1 ng); Wnt8 (100 pg);Dsh (1 ng); and β-catenin (200 pg) were added in the listed amounts.

When Wnt8 RNA was injected into a ventral vegetal blastomere at 4-8 cellstages, it induced an ectopic secondary axis. Co-injection of Wise RNAcompletely blocked Wnt8-induced secondary axis. This inhibition wascomparable to that mediated by a dominant negative form of Dsh (FIG.7C).

FIG. 7D shows that Wise functions extracellularly to block induction ofSiamois and Xnr3 by the Wnt pathway in ventral marginal zones. Wiseblocks the ability of Wnt8 to induce Siamois and Xnr3, but it does notinterfere with the ability of Dishevelled (Dsh) or β-catenin ((3-cat) toinduce these markers.

This inhibitory activity was examined at the molecular level in ventralmarginal zone explants by assaying for Wnt-dependent induction of Xnr3and Siamois, two immediate early response genes. In agreement with theaxial duplication assays, the induction of Xnr3 and Siamois in ventralmarginal zones by Wnt8 was blocked by the co-injection of Wise (FIG.7D). However, Wise had no effect on the ability of injectedintracellular components, such as Dishevelled and β-catenin to induceXnr3 and Siamois (FIG. 7D). This suggests that Wise functionsextracellularly to interfere with canonical Wnt signaling.

Example 10

The inhibitory effect of Wise on the Wnt pathway was further examined byassaying secondary head induction dependent upon simultaneously blockingboth BMP and Wnt signaling. When BMP signaling is blocked at the ventralmarginal zone by a truncated BMP receptor (tBR), an incomplete secondaryaxis is formed (FIG. 7E). However, simultaneous inhibition of both BMPand Wnt signaling resulted in the formation of a complete secondary axiswith eyes and cement glands. Co-injection of tBR and Wise induced acomplete secondary axis (FIG. 7F), demonstrating that Wise blocked theWnt pathway in this context.

Wise affected planar cell polarity. While the activation and inhibitionproperties of Wise in animal caps and embryos, described above, aredependent upon the canonical Wnt pathway, it is possible that Wise alsoinfluences the PCP pathway that branches at Dishevelled. Wnt11 isrequired for proper convergent extension movements of mesoderm duringgastrulation in frogs and fish, and this has been shown to be dependentupon the PCP pathway of Wnt signaling. Animal caps cultured in thepresence of Activin form mesoderm and undergo convergent extensionmovements, which can be blocked by reagents that either elevate ordecrease Wnt signaling. This implies that precise levels of Wntsignaling through the PCP pathway are essential for coordinated cellmovements. FIGS. 7E and 7F show that Wise acted as Wnt inhibitor andinduced head attribute formation in an incomplete secondary axis system.When BMP signaling was blocked at the ventral marginal zone by injectionof a truncated BMP receptor (tBR), an incomplete secondary axis wasformed (FIG. 7E). Co-injection of tBR and Wise induced a completesecondary axis with eyes (arrows) and cement gland (FIG. 7F).

FIGS. 7G-7I show how Wise blocks cell movements in Activin-treatedanimal caps. Control animal caps (FIG. 7G) undergo gastrulation-likemovements in the presence of Activin (FIG. 7H). In Wise injected animalcaps, elongation was blocked (FIG. 7I), but mesoderm induction occurred.In this animal cap assay, injection of Wise RNA blocked cell movementspreventing elongation of animal caps, but had no effect onActivin-induced mesoderm formation (FIGS. 7G-7I). This suggested thatWise influenced the Wnt-dependent PCP pathway, but whether activation orinhibition of the pathway resulted, cannot be distinguished. This effecton cell behavior in animal caps is consistent with and may explain thephenotypic effects observed in Wise-injected whole embryos. Wiseperturbed the morphogenesis of the neural tube, which failed to close.It was thickened and shorter, and there was a lateral expansion andbroadening of A-P markers. Many of these defects appear related toabnormal convergent extension movements during gastrulation. However,the fact that morpholino antisense oligo does not interfere the neuralA-P markers (FIG. 4), and that Wise is not predominantly expressed atgastrula state (FIG. 2), both suggest that endogenous Wise is unlikelyto be involved in gastrulation movement. Instead, Wise has a potentialto interfere with the Wnt-mediated PCP pathway.

Example 11

The mechanisms of action were investigated as potential physicalinteractions of Wise, with Wnt family members or their putativeco-receptors Frizzled (Hsieh et al., 1999) and LRP6 (Tamai et al., 2000)or Frizzled8 (Hsieh et al., 1999) with Wise conditioned medium, andassayed for interactions by immunoprecipitation (IP). In this assay,Wise bound to LRP6 and Frizzled 8, but not to Wnt8 (FIG. 8). Recentstudies have shown that individual members of the Dickkopf (Dkk) familyof secreted proteins can either antagonize or stimulate Wnt signalingthrough interaction with LRP6 (Brott and Sokol, 2002; Mao et al., 2001;Wu et al., 2000). Therefore, IP experiments were performed to determineif Wise shared common binding sites with Dkk1 or Wnt on LRP6. Theextracellular domain of LRP6 contains four EGF repeats and Dkk1interacts with repeats 3-4, while Wnt interactions seem to involverepeats 1-2 (Mao et al., 2001). It was found that Wise binds to LRP6 anda variant where EGF repeats 3 and 4 are deleted (ΔE3-4), but not to onein which EGF repeats 1 and 2 are removed (ΔE1-2) (FIG. 8A). Conversely,Dkk1 binds to LRP and ΔE1-2, but not to ΔE3-4 (FIG. 8A). These resultsshowed that Wise shared the domain on LRP6 essential for interactionwith Wnt and that Wise and Dkk1 modulate LRP6 activity by interactingthrough different domains. Wise and Wnt8 were tested to determinewhether they could bind to LRP6 at the same time, or whether theycompete for binding. As shown in FIG. 8C, Wise interferes with thebinding of Wnt8 to LRP6. This suggested a mechanism, whereby Wiseinhibits Wnt signaling by competing with Wnt8 for binding to LRP6 (FIG.8D).

In conclusion, the results demonstrate that Wise influenced both thecanonical and PCP pathways of the LRP/Wnt signaling cascade. The novelability to both activate and inhibit Wnt signaling through actions of asingle discrete regulatory molecule, places Wise in a unique position asa modulator of Wnt signaling.

Example 12

In this Example, Sost inhibition of the Wnt pathway is described. It hasbeen demonstrated that Wise acts to inhibit the Wnt pathway. Thefunctional inhibition of Wnt was shown to be derived from the secondexon of Wise, which encodes the cysteine knot. Since the cysteine knotof Sost is 70% homologous to that of Wise (FIG. 9), thus Sost'spotential functioning in a similar fashion was explored. Sost RNA waseither microinjected alone or in combination with other factors intoXenopus embryos and dorsal marginal zones were assayed for earlyimmediate Wnt response genes, Siamois and Xnr3 (FIG. 11). It was foundthat, like Wise, Sost was able to inhibit the action of Wnt on Siamoisand Xnr3 (FIG. 11). This Wnt inhibition by Sost was found to be workingupstream from β-Catenin (FIG. 11). Like Wise, Sost was able to rescuesecondary axis formation by Wnt (FIG. 11). However, unlike Wise, Sostwas unable to completely restore a normal axis (FIG. 11).

Wise has also been shown to induce En2 at a distance in Xenopus Nogginanimal cap assays. En2 expression at a distance is from an induction ofWnt gene activity. The conclusion was that Wise had induced, at adistance, more posterior neural markers in an anterior neuralized animalcap. Next it was analyzed whether Sost and Wise could be redundant bylooking to see if Sost could also induce En2, like Wise. Xenopus embryoswere either injected with Noggin and/or with Sost or Wise. We found thatWise injected animal caps induced En2 expression, however Sost injectedcaps did not (FIG. 11). This unexpected finding led to furtherexamination of these two genes.

Example 13

A Wise knockout mouse was made as detailed herein. A Neo-lacZ cassette,containing stop codons at the 3′ end, was inserted into a Wise gDNAsequence isolated bacterial artificial chromosome (BAC) from a 129strain of mouse by conventional cloning techniques. The mouse Wise DNAsequence is SEQ ID NO 1. A Sost knockout mouse can similarly be made.The mouse Sost sequence is SEQ ID NO 6. The Neo-lacZ cassette can beobtained from Stratagene (La Jolla, Calif.). The E. coli lacZ gene, whenintegrated into the mouse genome by standard cloning techniques, can beused as a reporter gene under the control of a given promoter/enhancerin a transgene expression cassette. The lacZ gene encodesβ-galactosidase, which catalyzes the cleavage of lactose to formgalactose and glucose. In the presence of X-gal chromogenic substrate,β-galactosidase converts the substrate, into an insoluble blue dye,allowing identification of cells containing lacZ activity.

The 129 mouse strain, commonly utilized in creating “knockout” mice, wasobtained from Jackson Laboratories, Bar Harbor, Me. The Wise knockoutmice produced lacked the presence of functional Wise polypeptidemolecules. Sost knockout mice are predicted to lack functional Sostpolypeptide molecules. Thus, these knockout mice may be referred to asfunctional mutants. In such mutant mice, protein translation isprematurely terminated.

A Neo-lacZ cassette, containing stop codons at the 3′ end, was insertedinto the first Exon of the Wise DNA, isolated BAC using the SmaI andEcoRI restriction sites. However, the Neo-lacZ cassette can also beinserted into a position within or adjacent to Exon 1 (SEQ ID NO 127)and Exon 2 (SEQ ID NO 128) of Wise. The Wise-containing BAC preparationwas exposed to cleavage enzymes, such as SpeI and BamHI, which yieldedhomologous arms containing 5′ UTR and 3′ intron nucleic acid sequences.These nucleic acid sequences permitted homologous recombination withwild type DNA from 129 mouse-derived embryonic stem (ES) cells uponintroduction of the BACs into ES cells by the electroporation methoddescribed in Example 32. The Neo-lacZ cassette contained one or morestop codons terminating translation of Wise polypeptide, leading toproduction of a truncated Wise polypeptide, which lacked the cysteineknot motif. The Wise cysteine knot region is significant because thisregion (1) is homologous to cysteine knot regions of Sost and otherfamily members as described herein, and (2) binds to LRP.

After recombination, the ES cells were grown in the presence of G148 forneomycin selection. Neo-lacZ cassette-containing ES cells wereneomycin-resistant and positively selected. There were three possibleevent outcomes occurring when the resultant ES cells were cultured inneomycin-containing media: First, Wise Neo-lacZ cassette-containing EScells grew, indicating a successful homologous recombination eventwithin the first Exon region of Wise, as predicted. Second, norecombination occurred, resulting in the lack of the presence of aprotective Neo-gene in the ES cells and cell death. Third, recombinationoccurred outside the first Exon of Wise, conferring neomycin resistanceand ES cell survival and growth.

To distinguish between the above first and third categories ofrecombination events in live neomycin-resistant ES cell cultures,genomic DNA (gDNA) extracted from ES cells was divided into twoaliquots. One part was frozen (−20 deg. C) for further investigation,and the other part was digested in vitro with EcoR I for Southern Blotanalysis. By using a 3′ probe within Wise Exon 2, EcoR I digestionyielded either a 6.8 Kb fragment associated with a homologousrecombination event, or a 9.0 Kb fragment associated with a randomintegration event. Frozen cultures from those plates that exhibitedhomologous recombination event were thawed, expanded and furtherprocessed for creation of Wise mutant mice by micro-injection of theseWise Neo-lacZ cassette-containing ES cells into blastomeres as describedhereinafter.

In the process of electroporation of mouse ES cells, linearized Wisenucleic acid sequences containing the Neo-lacZ cassette were insertedinto the nuclei of ES cells for incorporation into the host ES cell DNA.Similarly, Sost nucleic acid sequences with the Neo-lacZ cassette can beinserted into the nuclei of ES cells for incorporation into other hostES cell DNA. The electroporation process steps were as follows. ES cellswere obtained from removed blastocysts obtained from mouse uteri andgrown on mitotically inactivated Mouse Embryonic Fibroblast (MEF) feederlayers. An ES cell frozen ampoule was thawed and transferred to asterile dish containing MEFs as a feeder layer at a concentration of1×10⁶ cells per 10 centimeter (cm) dish. ES cells were grown on the MEFfeeder layer in ES media in T-150 flasks. ES cells were centrifuged andwashed in transfection buffer (1×Hebs). ES cells were then placed in asterile “flat pack” 1.8 mm gap cuvette (BTX order #485), and the cuvettewas inserted between the safety stand contacts.

The power was switched to the on position with the BTX 600 or equivalentelectroporator set to 500V/capacitance and resistance, 500 uFcapacitance timing, 360 ohms R8 resistance timing, and charging voltage185V. After pipetting the ES cells up and down with a 5 ml pipette,targeting construct DNA (40 μg of clean linear DNA in 1×TE @ 1 μg/μl foreach electroporation) was added to the ES cells in a microfuge tube.Cells were pipetted up and down gently with a Pasteur pipette. Cellswere slowly added to the cuvette which was then placed into theelectroporation chamber. The start button was pushed, andelectroporation occurred. After completion, electroporated ES cells wereremoved from the cuvette and placed in 5.0 ml of fresh ES medium in acentrifuge tube. 2 ml of transfected ES cells were added to each dishcontaining inactivated MEF feeder layers. Dishes, were rocked slowly toevenly disperse cells and incubated. ES cells were fed on day 9 and 12with selection medium, and clones became visible as small nests under aninverted microscope. Clones were picked on day 13 or 14 using a pipettorset between 30 and 50 μL Clones were each placed into one of 24 wellscontaining ES selection medium. On day 16 or 17, clones were frozen inES freezing medium and stored at −70° C.

Each set of ES cells containing mutant Wise genes were injected intomouse embryos for creation of transgenic “knockout” mice. Such ES cellswere microinjected into early mouse embryos (i.e., blastocysts) whichwere then transferred to surrogate mothers for embryonic development.Targeted stem cells containing mutant Wise were placed in an injectionchamber with expanded blastocysts. Stem cells were loaded into theinjection needle and inserted into the blastocoel cavity of therecipient 129 or C57BL/6 embryo, then implanted into the uterus of afoster mother. Chimeric offspring were identified by coat color (i.e.,at 2 weeks) or other markers and confirmed by Southern blot analysis oftail biopsies (i.e., at 3 weeks). Similarly, ES cells containing mutantSost genes can be made and injected into ES cells to make Sost knockoutmouse embryos.

The resulting pups (i.e., chimeras) contained a (+) gene in some cellsand a (−) gene in other cells. Chimeras were mated with normal mice.Pups were identified that carry one (+) and one (−) copy of the Wisegene, and these animals were mated with each other.

The mouse pups were then analyzed. About 25′ percent of the pups werefound to have inherited the (−) gene from both parents and completelylack the (+) or wild type gene. Homozygous (−) gene pups lacking theWise wild type gene were termed “Wise knockout mice.” Similarly,homozygous (−) gene pups lacking the Sost wild type gene can be made,and these are referred to as “Sost knockout mice.” Wise knockout micewere then utilized for subsequent experiments to determine effectsrelating to bone mineral density, bone deposition, embryo implantation,hair development, tooth abnormalities, ophthalmic abnormalities. Sostknockout mice may similarly be made and utilized in phenotypicexperiments.

Example 14

A Sost knockout mouse can be made using the procedure of Example 13above. Briefly, a Neo-lacZ cassette, containing stop codons, can beinserted into a Sost gDNA isolated BAC from a 129 mouse strain byconventional cloning techniques. The Sost-containing BAC preparation canbe electroporated and allowed to undergo homologous recombination intoES cells and be exposed to selection. ES cells containing mutant Sostcan be injected into mouse embryos for creation of transgenic Sostknockout mice as previously described.

Example 15

In this Example, the Wise knockout mice, produced in Example 12, wereused to investigate the effect of the absence of a functional Wisepolypeptide molecule upon opthalmic development. It was determined thatophthalmic abnormalities developed in these mutant mice. Immunodetectionof Wise protein production in murine retinal regions was used todetermine the efficacy of induced Wise mutation in the Wise mutant mice.

Polyclonal anti-Wise peptide antibody was prepared by rabbitimmunization with Wise peptide antigens. Such antibodies were directedagainst the cysteine knot loop encoded by Exon 2 of Wise.

Zymed FITC-conjugated secondary polyclonal antibody directed againstprimary rabbit anti-Wise peptide antibody was also utilized in ahistological sandwich immunoassay. Eye mounts containing retinas orsections were stained with anti-Wise antibody and FITC-conjugated secondantibody. In wild type mice, anti-Wise reactivity was detected assecreted Wise protein in the ganglion cell and optic fiber layers and inrods and cones. However, Wise mutant mice eyes lacked detectableanti-Wise peptide reactivity, indicating absence of Wise from tissues ofthese mutant mice.

The Wise mutant mice appeared to have lost the majority of the opticnerve fibers and had increased rod and cone layers in the retina (FIG.12). These mice also exhibited abnormal retinal ganglion cells. Wiseprotein was found in the inner plexiform layer, ganglion cells andfibers, and in the rods and cone layer of a 2.5 month mouse retina (FIG.12). Unlike Wise, Sost was found in the tissues adjacent to theneuroepithelium of the diencephalon at E18 dpc.

Example 16

Wise mutants were analyzed to compare BMD in Wise mutants as compared tothat in wild type mice. A Piximus instrument (Faxitron) was used tomeasure BMD, computed in whole mice by measurement of bone weightdivided by area of bone measured.

The BMD in Wise mutants from the C57BL6 and 129 mouse strains wascompared with that in wild type (wt) mice by the student t-test method.The resultant p value obtained for the BMD differences between C57BL6vs. Wise mice was 0.0017. This indicates that BMD values increased inWise mutant mice as compared to C57BL6 wt mice, with a significantdifference between groups (p<0.01) observed. Increased BMD values werealso observed in the 129 Wise mutant mice in comparison with 129 wtmice.

Related to this finding, FIG. 13 shows results of bone staining and BMDmeasurements. FIGS. 13A and 13C depict hematoxylin and eosin (H&E)staining of cross-sections of bone tissue from 16 to 18 days post cortum(DPC) mice. FIGS. 13B and 13D show the same bone regions as FIGS. 13Aand 13C; however, FIG. 13B shows staining with S-35 radiolabel attachedto Sost RNA probes, wherein Sost is located in osteoblasts in 16 to 18DPC mice. FIG. 13D also shows staining with anti-Wise peptide primaryantibody and FITC-conjugated secondary antibody, and localization ofWise in hypertrophic and prehypertrophic proliferating chondrocytes.

FIGS. 13E and 13F show graphical depictions of bone density measurementsand total bone weight measurements, respectively. FIG. 13E shows thatobservable significant differences in BMD measurements between Wisemutant and wild type mice occur at ages between 0 and 3 months. Wisemutant bone is higher in density than wt bone in this age range. At 4months, there appears to be no significant difference between mutant andwt groups. FIG. 13F depicts total bone weight measurements. Note that at2.5 months wt bone weight is 19.87, significantly different from theWise bone weight of 24.67. Therefore, some of the increase in BMD foundat 2.5 months can be attributed to increase bone weight and notnecessarily an increased BMD. Consistent with data in FIGS. 5E and 5F,it is concluded that during the 0 to 3 month period, bone depositionoccurs. However, at the 4 month maturation stage, it is postulated thatregulatory genes are switched on to remodel bone deposition and boneremoval, wherein osteoclasts may be triggered to remove previouslydeposited bone.

In summary, one tissue cell type that both Sost and Wise genes appear toaffect in a similar fashion is the bone. Sost is expressed inosteoblasts. Sost may also be expressed in osteoclasts. In contrast,Wise is expressed in periosteum, and its protein is found onchondrocytes (proliferating, prehypertrophic and hypertrophic), but notin the growth plate (FIG. 5). Yet, both Sost and Wise genes display asimilar phenotype of increased bone density, albeit potentiallyactivated at different developmental stages. As such, Wise mutant micehave increased bone density during early prenatal bone development(under 4 months), and cease to exhibit increased bone density oncebone-modeling begins (4 months; FIG. 5). However, Sost mutations resultin increased bone density during the subsequence developmental stage inwhich the adult bone remodeling process occurs.

Example 17

Genetic regulation in tooth and jaw development was examined in wildtype and Wise mutant mice as shown in FIG. 14. The mice were dissected,and the jaws were placed in a proteinase K solution (2×SSC, 0.2% SDS, 10mM EDTA, and 100 ul of 10 mg/ml proteinase K) overnight at 55° C. Thenext day the jaws were air-dried. A digital Faxitron was used forcapturing X-ray images of the mouse jaw. The teeth were removed usingtweezers.

FIGS. 14A, 14D, and 14G show hematoxylin and eosin staining of a jawcross-section. FIGS. 14B, 14E, and 14H show S-35 RNA probe-labeled Soststaining. FIGS. 14C, 14F, and 14I show S-35 RNA probe-labeled Wisestaining. Generally, these figures show that Sost appears inondontoblasts and osteoblasts. In contrast, Wise is found in incisors,dental follicles, and hair follicles in the whisker pad.

The top sectional FIGS. 14A,14B, and 14C show a bilateral view of twomolars with developing tooth buds. FIG. 14C shows that Wise labelslayers of the dental follicle of molar teeth.

The middle sectional FIGS. 14D, 14E, and 14F show a molar tooth bud at ahigher magnification. FIG. 14E shows Sost staining in osteoblasts of thetrabecular bone adjacent to the molar tooth. Visible staining of theodontoblasts occurs along the base of each molar. FIG. 14F shows Wisestaining of dental follicle layers.

The bottom sectional FIGS. 14G, 14H, and 14I show incisor tooth stainingpatterns. FIG. 14G shows the morphological features of two incisors,with the nasal cleft between them, tongue, and hair follicles of thewhisker pad. FIG. 14H shows Sost staining in osteoblasts of trabecularbone. FIG. 14I shows prominent Wise staining of incisors. Hair folliclesand the whisker pad are also stained with Wise labeled RNA probes.

FIGS. 14J and 14K show X-ray photographs of incisor teeth in the maxilla(upper jaw) regions of the wild type and Wise mutant mice, respectively,utilizing a 129 strain genetic background. The Wise mutant jaw, shown inFIG. 14K, possesses an additional incisor tooth (i′) not present in thewt mouse shown in FIG. 14J. The additional tooth may originate fromeither an additional tooth bud or, alternatively, from a bifurcation ofthe original incisor.

FIGS. 14L, 14M, 14N, and 14O show the patterning in molar teeth observedin wt (FIGS. 14L and 14N) as compared to Wise mutant mice (FIGS. 14M and14O), against a C57BL6 genetic background (FIGS. 14L and 14M) and 129background (FIGS. 14N and 14O). FIG. M shows an additional M1 molar inthe Wise mutant mouse in comparison to the M1, M2, and M3 molars presentin the wt mouse in FIG. 14L. FIG. 14O shows tooth abnormalities in theWise mutant mouse. The M1 and M2 molar teeth are fused together.Moreover, there is a reversal of the order of molar bone patterning,wherein an M3/M2-1 pattern appears in the Wise mutant, in contrast tothe wild type's M1/M2/M3 pattern. Occasionally, an additional M4 molartooth appears in the Wise mutant.

It was observed that Wise mutant mice possessed tooth abnormalities. Theincisors occurred in duplicate number in comparison with wt mice, andthese teeth required weekly clipping from the weaning stage onwards. Inaddition, the molars also displayed abnormal patterning. The threemolars were often found in reverse orientation and also showed fusion ofM1 and M2. In contrast to Wise mice, Sost human mutations did notdisplay these molar and incisor tooth phenotypic abnormalities, probablybecause of the differences in Sost and Wise gene expressiondistributions in bone. Thus, Sost was expressed in the polarizedodontoblasts and the surrounding osteoblasts. Wise, on the other hand,is expressed in the dental follicle surrounding the tooth bud and in theincisors. Thus, Sost and Wise were expressed in complementary celltypes, wherein differing tooth and eye phenotypic expression patternsare anticipated and observed in Sost human mutations and Wise mutants.

Example 18

Plasmid vectors containing Wise nucleic acid sequences were prepared forthe purpose of producing Wise proteins and polypeptides. Sost vectorswere similarly prepared for the purpose of producing Sost proteins andpolypeptides. The expression vector, pET-28b (Novagen pET SystemManual), was used for the expression of Wise and Sost, LRP5 and LRP6sequences. This plasmid utilizes the phage T7φ10 gene promotor. Thispromotor is not recognized by E. coli DNA dependent RNA polymerase, andthus will not produce substantial levels of the polypeptide unless T7RNA polymerase is present. Strain BL21 (DE3) contains a lysogenic phagethat encodes the required polymerase under control of the lacUV5promotor. A recombinant protein that was made was the intact Wise, Sost,LRP5 or LRP6 proteins. The Wise pET vector which was created by placingan EcoRI-HindIII fragment containing chick Wise cDNA into the pET28Bvector which was then digested with EcoRI-HindIII. Extra amino acids 5′to Wise Start, ATG were removed, along with extra amino acids 3′ to theWise stop codon. The Sost pET vector was created by placing a BamHI-XhoIfragment containing mouse Sost cDNA into pET BamHI-XhoI. The amino acidsfrom the 5′ and 3′ ends to the Sost coding region were removed usingmutagenesis. The 3′ amino acids were deleted and the missing ELENAY wasinserted at the 3′ end.

Example 19

In this Example, the method used for protein production for Wise, Sost,LRP5, and LRP6 polypeptides in HEK293 mammalian cells is outlinedbriefly. PCS2+ Sost-FLAG, PCS2+Wise-FLAG, or PCS2+LRP6 IgG,PCS2+LRP5-Myc DNA was transfected into the HEK293 cells using FuGENE 6Transfection Reagent (10 μg DNA/100 mm plate) (Roche Diagnostics Corp.,Indianapolis, Ind.). The FuGENE reagent is a multi-component lipid-basedtransfection reagent that complexes with and transports DNA into thecell during transfection. Adherent cells were plated one day beforetransfection, and freshly passaged HEK293 suspension cells wereprepared. FuGENE 6 reagent:DNA ratios of 3:2, 3:1 and 6:1 were used totransfect HEK293 suspension cells.

After incubation, cell supernatants, containing the polypeptide ofinterest (Wise, Sost, LRP5, LRP6), were collected on days 1, 2, 3, and4. Polypeptide-containing supernatants were concentrated by AmiconUltra-15 column passage (20 ml to 500 μl). Some aliquots were frozen,and other aliquots were used in Western blot and immunoprecipitationquantitation and characterizations using standard methodologies.Mixtures of Wise and LRP5, Wise and LRP6, Sost and LRP5, and Sost andLRP6 were analyzed for binding by immunoprecipitation and Western blotanalysis. See SuperSignal West Dura Western Blotting Kit (Pierce,Rockford, Ill.), Trans-Blot SD Semi-Dry Electrophoretic Transfer andMini-PROTEAN 3 Electrophoresis (Bio-Rad Labs., Richmond, Calif.),Hybond-P PVDF Membrane for protein transfer (Amersham PharmaciaBiotech), Chroma Spin Columns (Clontech, Palo Alto, Calif.).

Immunoprecipitation was performed with anti-Wise antibody, anti-Myc,anti-Flag, and protein G sepharose (Sigma, St. Louis, Mo.) or protein Asepharose (Repligen). Briefly, transfected cell supernatents wereprepared and 1-3 μg of antibody added. After incubation, 30 μl ofprotein G sepharose was added, incubated, and beads were centrifuged.Beads, containing antibody from supernatents as the immunoprecipitate,were washed in buffer, then submitted to SDS-PAGE analysis and WesternBlot analysis. Alternatively, immobilized antibody was used inimmunoprecipitation of proteins.

In Western Blots, electrophoresis was performed upon the cellsupernatent material above. After wash, water rinse, and equilibrationof the PVDF membrane in transfer buffer, papers were sandwiched asfollows: pre-soaked thick paper, membrane, gel, pre-soaked thick paper.Power was turned to 10V to 15V for 30 min. After transfer of protein tothe HyBond-P PVDF membrane, the membrane was incubated in blockingbuffer, rinsed, and incubated with antibody solution. After wash, asecondary antibody was added, washed, then ECL-plus added. Afterexposure of X-ray film, patterns were read. As such, protein productionin PCS2+ transfected HEK293 cells was performed to support purificationand characterization of Wise, Sost, LRP5, and LRP6 polypeptides.

Example 20

A method for the production of large quantities of Wise and Sostpolypeptides is described. Bacteria cells transfected with either theWise or Sost genes can be grown. E. coli strain DME558 is grown on LBagar plates at 37° C.

For P1 transduction, a P1 viral lysate of the E. coli strain DME558 isused to transduce a tetracycline resistance marker to strain BRE51(Bremer, E., et al., FEMS Microbiol. Lett. 33:173-178 (1986)) in whichthe entire OmpA gene is deleted (Silhavy, T. J., et al., Experimentswith Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y. (1984)). Strain DME558, containing the tetracycline resistancemarker in close proximity of the OmpA gene, is grown in LB medium untilit reached a density of approximately 0.6 OD at 600 nm. One tenth of amilliliter of 0.5 M CaCl₂ is added to the 10 ml culture and 0.1 ml of asolution containing 1×10⁹ PFU of PI_(vir).

The culture is incubated for 3 hours at 37° C. After this time, thebacterial cell density is visibly reduced. 0.5 ml of chloroform is addedand the phage culture is stored at 4° C. Because typically 1-2% of theE. coli chromosome can be packaged in each phage, the number of phagegenerated covers the entire bacterial host chromosome, including thetetracycline resistance marker close to the OmpA gene.

Next, strain BRE51, which lacks the OmpA gene, can be grown in LB mediumovernight at 37° C. The overnight culture is diluted 1:50 into fresh LBand grown for 2 hr. The cells are removed by centrifugation andresuspended in MC salts. 0.1 ml of the bacterial cells are mixed with0.05 of the phage lysate described above and incubated for 20 min. atroom temperature. Thereafter, an equal volume of 1 M sodium citrate isadded and the bacterial cells are plated out onto LB plates containing12.5 μg/ml of tetracycline. The plates are incubated overnight at 37° C.Tetracycline resistant (12 μg/ml) transductants are screened for lack ofOmpA protein expression by SDS-PAGE and Western Blot analysis, asdescribed below. The bacteria resistant to the antibiotic possess thetetracycline resistance gene integrated into the chromosome very nearwhere the OmpA gene had been deleted from this strain. One particularstrain was designated BRE-T^(R).

A second round of phage production can be then carried out with thestrain BRE-T^(R), using the same method as described above.Representatives of this phage population contain both the tetracyclineresistance gene and the OmpA deletion. These phage are then collectedand stored. These phage are used to infect E. coli BL21(DE3). Afterinfection, the bacteria contain the tetracycline resistance marker. Inaddition, there is a high probability that the OmpA deletion is selectedon the LB plates containing tetracycline.

Colonies of bacteria obtained from plates are grown up separately in LBmedium and tested for the presence of the Wise and Sost protein and OmpAprotein as judged by antibody reactivity on SDS-PAGE western blots.

The SDS-PAGE is a variation of Laemmli's method (Laemmli, U. K., Nature227:680-685 (1970)) as described previously (Blake and Gotschlich, J.Exp. Med. 159:452-462 (1984)). Electrophoretic transfer to Immobilon P(Millipore Corp. Bedford, Mass.) is performed according to the methodsof Towbin et al. (Towbin, H., et al., Proc. Natl. Acad. Sci. USA76:4350-4354 (1979)) with the exception that the paper is first wettedin methanol. The Western blots are probed with phosphatase conjugatedreagents (Blake, M. S., et al., Analyt. Biochem. 136:175-179 (1984)).

Example 21

The fusion constructs of Example 18 can be used to transform theexpression strain BL21 (DE3) ΔOmpA of Example 19. The transformationplates are cultured at 30° C. Colonies of both types are isolated fromthese plates and analyzed. It is generally found that virtually alltransformants contained the desired plasmid DNA.

Various fusion-Wise clones are then analyzed for protein expression. Theclones are induced and grown in LB media containing 0.4% glucose and 118μM carbenicillin instead of ampicillin with an aeration speed of 100 to150 rpm and at about 30° C. The expression of the Wise protein isanalyzed by loading 0.1 ml of the culture of total E. coli proteins onan 8-16% gradient SDS gel.

E. coli strain BL21 (DE3) ΔOmpA [pNV-3] can be grown to mid-log phase(OD=0.6 at 600 nm) in Luria broth. Isopropyl thiogalactoside is thenadded (0.4 mM final) and the cells were grown an additional three hoursat 30° C. The cells are then harvested and washed with several volumesof TEN buffer (50 mM Tris-HCl, 0.2 M NaCl, 10 mM EDTA, pH=8.0) and thecell paste stored frozen at −75° C.

For purification, about 3 grams of cells are thawed and suspended in 9ml of TEN buffer. Lysozyme was added (Sigma, 0.25 mg/ml) deoxycholate(Sigma, 1.3 mg/ml) plus PMSF (Sigma, 10 μg/ml) and the mixture wasgently shaken for one hour at room temperature. During this time, thecells lyse and release DNA causing the solution to become viscous. DNaseis then added (Sigma, 2 μg/ml) and the solution again mixed for one hourat room temperature. The mixture is then centrifuged at 15 K rpm in anSA-600 rotor for 30 minutes and the supernatant discarded. The pellet istwice suspended in 10 ml of TEN buffer and the supernatants discarded.The pellet is suspended in 10 ml of 8 M urea (Pierce) in TEN buffer.

Alternatively, the pellet can be suspended in 10 ml of 6 M guanidine HCl(Sigma) in TEN buffer. The mixture is gently stirred to break up anyclumps. The suspension is sonicated for 20 minutes or until an evensuspension is achieved. 10 ml of a 10% aqueous solution of3,14-ZWITTERGENT is added and the solution is thoroughly mixed. Thesolution is again sonicated for 10 minutes. Any residual insolublematerial is removed by centrifugation.

This mixture is then applied to a 180.times.2.5 centimeter (cm) columnof Sephacryl-300 (Pharmacia) equilibrated in 100 mM Tris-HCl, 1 M NaCl,10 mM EDTA, 20 mM CaCl₂, 0.05% 3,14-ZWITTERGENT, pH=8.0. The flow rateis maintained at 1 ml/min. Fractions of 10 ml are collected. Threedimensional conformation was restored in Wise during the gel filtration.The absorbance (OD=280 nm) of each fraction is measured and thosefractions containing protein are subjected to SDS gel electrophoresisassay for Wise. Those fractions containing Wise are pooled and stored at4° C. for 3 weeks. During the incubation at 4° C., a slow conformationalchange occurs. The Wise protein remained in solution without theelevated levels of salt. The pooled fractions are then dialyzed against50 mM Tris-HCl, 200 mM NaCl, 10 mM EDTA, 0.05% 3,14-ZWITTERGENT, pH=8.0.This material is applied to a 2.5× cm Fast Flow Q Pharmacia columnequilibrated in the same buffer. Any unbound protein is eluted withstarting buffer. A linear 0.2 to 2.0 M NaCl gradient is then applied tothe column. The Wise elution profile can be characterized. Fractions areassayed by SDS-PAGE and the purest fractions pooled and dialyzed againstTEN buffer containing 0.05% 3,14-ZWITTERGENT. Thus, cells transfectedwith the constructs can be isolated for Wise protein production.Similarly, Sost transfected cells can be isolated for Sost proteinproduction.

Example 22

The family tree associations of relatedness between Sost, Wise, andother cysteine knot proteins were analyzed. Sost and Wise cysteine knotprotein sequences were analyzed using BLAST, and all significantsequences were isolated. The cysteine knots from all sequences werealigned using the software T-Coffee and then analyzed with Phylipbootstrap neighbor joining methods. To determine chromosomal locations,Wise and Sost DNA sequences were compared against sequences in the mouseand Ensembl database (http://www.ensembl.org/Mus_musculus/blastview).

The BLAST program optionally filters out low-complexity regions from thesearch and assigns scores with well-defined statistical interpretationsuch that real matches of related sequences can be distinguished fromrandom background hits. The default scoring matrix is BLOSUM62. Thesignificance of each of the matches is given an Expect (E) score,defined as the expected number of alignments between a random querysequence and a database of random sequences of the same “effective”length and number that will score as well.

A Wise cDNA, (SEQ ID NO 1) was isolated and submitted to NCBI for BLASTsequencing. The Wise cDNA was comprised of 618 nucleic acids,corresponding to 206 amino acids in the wild type polypeptide molecule.743,070 sequences were searched in the database. From this search, itwas determined that Wise and Sost were related. Both genes had two exonsand an intron. Exon 2 for both genes was 400 bp long and possessed twocysteine domains that were 70% identical.

FIG. 9D shows the family tree relatedness between cysteine knot proteinmembers Sost and Wise. In initial experiments, when the BLAST analysiswas performed for Wise protein alone, only CCN family members (e.g.,Slit, Mucin) were obtained as related family members. When Sost alonewas run, only DAN family members (e.g., Caronte, Gremlin) weredetermined to be related. However, when Sost and Wise BLAST analyseswere performed together, the family tree was that depicted in FIG. 9D.In this analysis of cysteine knot protein relatedness, it was noted thatthe DAN family had only one cysteine knot motif. In contrast, the CCNfamily and Slit and Mucin family possessed ten different protein motifs.Other family proteins had an additional cysteine knot moiety. G and Pare conserved.

In FIG. 9D, the red dots indicate significant relatedness among cysteineknot proteins. Thus, FIG. 9D depicts the following family branchassociations, wherein Sost and Wise are present in one branch. Nov,CTGF, Cyr61, and Cef10 are present in one closely related branch to Sostand Wise. Cerberus, Caronte, and Gremlin are in a second closely relatedbranch. The aforementioned three branches are more remotely related tothe following branches: the Muc2, Apomucpig, and Muc58 branch; the VWFbranch, and the Slit 1, Slit2, Slit3, Muc5AC, and Gastmuc branch.Numerical values in the tree in FIG. 9D indicate a measure of thesignificance of protein associations. The closer a number is to 100, themore significant the association. Numerical values of less than 50indicates insignificant associations. Thus, the numerical value of 97between Sost and Wise is highly significant.

Example 23

An in situ protocol for detection of gene expression in Sost mutant micewas conducted. 3′ untranslated regions of the Sost gene were obtained.

DNA from these 3′ translated gene regions were linearized from thevector, then clipped at the 5′ end. Subsequently, this sequence wastranscribed to produce an antisense RNA molecule. The antisense RNAmolecule was labeled with a deoxygenin (DIG) substrate tag. TheDIG-labeled RNA was then utilized to bind to an embryo's RNA.

In preparation for staining, a whole embryo was dehydrated and thenbleached at the pigment stage. The next day, the embryo was washed andtreated with detergent to induce permeability in subsequent staining.When the DIG-labeled RNA was incubated with RNA from an embryo, apurple-blue color was developed in whole embryo staining in the presenceof alkaline phosphatase, NBT and BCIP. Using this procedure, Sostexpression in whole embryo tissue was characterized.

Example 24

A chick Wise pET28b vector was made. The Novagen pET28b(+) vectors usedcontained fl origin, N-terminal histidine, T7, and optional C-terminalhistidine tags. Single-stranded sequencing was performed using the T7terminator primer. An EcoR I-HinD III nucleic acid fragment was obtainedfrom a chick Wise-containing pcDNA3.1-Myc-His vector for insertion intothe pET28b vector by established Novagen methods (Novagen, Madison,Wis.) described in various pET28b examples herein.

Example 25

A mouse Sost pET28b vector was made. A Sost-V5 epitope-tagged versionwas utilized as a base construct for making the pET28b(+) construct.Subsequently, Sost was removed from the base construct using BamHI andXhoI enzymes and inserted into pET28b vectors according to Novagenmethods as previously described. The Sost-containing preparation wasexpanded using the PCR method. Nucleic acids encoding thirteen excessamino acids were removed 5′ from the start codon of the Sost nucleicacid sequence utilizing the Stratagene site-directed mutagenesis kit.Also removed were extra restriction enzyme sites adjacent to Xho orlocated at the 3′ end of Sost. Naturally occurring nucleic acids in Sostencoding the last six ELENAY amino acids were added using mutagenesis.

Example 26

In this Example, the chick Wise-FLAG sequence was inserted into thepCS2+ vector by procedures discussed in Example 19. The chick Wisesequence was placed in the pCS2+ vector using the EcoRI and SpeI/XbaInucleases by cloning. The pCS2+ vector also contained T7, ClaI, BamHI,Sp6, and CMV sites. The chick Wise polypeptide was expressed and used todetermine binding to LRP and BMPs.

Example 27

This Example briefly describes the insertion of a mouse Sost sequenceinto the pcDNA3.1/V5-His-TOPO® vector. This TOPO® vector includes a CMVpromoter, T7 promoter/priming site, multiple cloning site, V5 epitope,polyhistidine tag, SV40 promoter, neomycin resistance gene, andampicillin resistance gene. Mutagenesis permitted creation of the wildtype Sost-V5 vector using the following steps: (1) addition of thesequence encoding six ELENAY amino acids, and (2) addition of the EcoR Isite to the 5′ end of the Sost sequence.

Example 28

Human wild type LRP6 and mutant LRP6-Δ3,4 gene constructs in the pET28bvector were created and characterized. These gene constructs can then beutilized for the production of the corresponding mutant LRP6-Δ3,4protein molecule. After cloning the foregoing LRP6 gene construct intothe pET28b vector by the method previously described in Example 3, thepET28b vector DNA was digested with BamHI and XhoI enzymes to yield theLRP6 sequence in soluble form for further characterization. This nucleicacid sequence was not linked to the transmembrane.

The EcoRV site was then mutated within the vector backbone using theStratagene II QuikChange XL-Site Directed mutagenesis kit. This kit'sprocedure is used to make point mutations, amino acid substitutions,frame shift mutations, or insertion of single or multiple adjacent aminoacids in Wise and Sost genes that encode polypeptides. The pET28 vectorwas digested with XhoI and EcoRV. The purified BamHI/EcoRV restrictionenzyme fragment was cloned into pET28b. The first band corresponded toEGF1,2; and the second band corresponded to EGF3,4. The LRP6-derivedEGF1,2 fragment was cloned into the pET28b vector containing BamHI andEcoRI sites by homologous recombination as previously described inExample 13. The Stratagene mutagenesis kit was used to obtain mutationsin the pET28b vector containing the LRP6-derived EGF1,2 sequence.Subsequently, XhoI, NotI and EcoRV sites were introduced into themultiple cloning site of the pET28b vector. These sites permittedopening of the circular nucleic acid sequence with EcoRV and BamHendonucleases to allow insertion of the LRP6-derived EGF1,2 fragmentinto the pET28b vector. LRP6-Δ3,4 protein was then expressed from thepET28b vector.

Example 29

This Example relates to the creation of the human LRP5Δ3,4mutant-containing pET28b vector. Similarly, an LRP5 Δ-4mutant-containing pET-28b vector can be made. A human LRP5 nucleic acidsequence inserted into the CS2+ vector was obtained. The coding sequencefor LRP5 in this vector runs from the EcoRI site to the XbaI site. Theintact LRP5 was obtained by digestion with EcoR I and Xba I nucleases.As in Example 18, the purified BamHI/XbaI fragment was then digestedwith the XhoI enzyme to yield two bands, corresponding to LRP5 EGF1,2and EGF3,4 fragments. As previously, the LRP5 EGF1,2 sequence wasinserted into the p28b vector containing EcoRI and XhoI sites.Site-directed mutagenesis was used to (1) remove the stop codon 5′ tothe actual start site and (2) delete extraneous nucleic acids located 5′to the start of the LRP sequence. The LRP Δ3-4 and LRP Δ3-4 vectorsubsequently can be used to independently transfect E. coli cells forproduction of LRP Δ3-4 and LRP Δ3-4 polypeptide molecules respectively.

Example 30

This Example relates to the creation of the secreted LRP5-myc CS2+vector. The human LRP5 containing pCS2 vector was obtained. Stratagensite-directed mutagenesis resulted in the following sequencemodifications: (1) addition of the Myc tag upstream of the transmembranedomain, and (2) addition of XbaI and XhoI sites flanking the Myc tagregion. Removal of the LRP5 sequence encoding the region that tethersthe protein to the membrane was performed by digestion with XbaInuclease. The resultant religated nucleic acid sequence encoded asecreted form of LRP5, lacking the tethered portion.

Example 31

Hybridoma cell lines were prepared that can secrete monoclonalantibodies reactive with Wise wild type proteins, polypeptides, wholemolecules, and fragments. The technology for producing monoclonalantibodies is well known. See generally E. A. Lerner, “How To Make AHybridoma”, Yale J. Biol. Med., 54, pp. 387-402 (1981); M. L. Gefter etal., “A Simple Method For Polythylene Glycol-Promoted Hybridization OfMouse Myeloma Cells”, Somatic Cell Genet., 3, pp. 231-36 (1977).Briefly, murine X63AG8.653 myeloma cells are fused to lymphocytesisolated from spleens of mice immunized with a preparation comprising ofWise polypeptide (e.g., wild type Wise polypeptide SEQ ID NOS 45,114-119), and the culture supernatants of the resulting hybridoma cellsare screened as described herein for anti-Wise antibody bindingactivity. The myeloma cell line is HAT-sensitive, wherein growth in HATmedium selects for growth of HAT-resistant hybridoma cells.

To prepare Wise protein Immunogen, KLH-Immunogen is made. Wise Immunogenmay be derived from Wise proteins or polypeptides. Representative Wisewild type proteins and polypeptides are SEQ ID NOs 45, 52, 104-106, andrepresentative Wise mutant polypeptides are SEQ ID NOs 114-119. EachBalb/c mouse is immunized subcutaneously with 0.2 ml of a preparationcontaining about 100 μg of Wise polypeptide in PBS (“Immunogen”) mixed1:1 with Complete Freund's Adjuvant (CFA). Wise polypeptide was producedaccording to the method described in Examples 20-21. The Wise Immunogenpolypeptide can be derived from wild type Wise molecules, asspecifically described in SEQ ID NOs 45, 52, 104-106, 114-119. ShortenedWise polypeptide molecules are SEQ ID NOs 115-119. Three days after thefinal booster injection, mice are exsanguinated, antisera titrated, andisolated spleen cells are fused with the non-secreting mouse myelomacell line, SP2/0 Ag 14 (ATCC Designation CRL 8287). Thielmans, K., etal., J. Immunol. 133:495 (1984).

Prior to fusing, the resultant mouse antisera are titered to determinethe concentration of anti-Wise antibodies made by each mouse. Pre-immunesera noted above are diluted in the same manner as the immune sera andused as controls. Microtiter wells are coated with 1.5 μg of BSA-Wiseantigen prepared by incubating bovine serum albumin (BSA fromCalbiochem, Catalog #12657, as described by Makita et al., J. Biol.Chem., 267(8), pp. 5133-5138 (1992). The antigen coated wells are sealedwith Mylar sealing tape (Corning) and incubated overnight at 4° C. Themicrotiter plates are subsequently washed and blocked in aBSA-containing solution. After incubation, the microtiter plates arewashed and 100 μl of a goat anti-mouse IgG (gamma chain specific)horseradish peroxidase-conjugated antibody (Sigma) is added to all wellsand incubated. Ortho-phenylenediamine (OPD) Peroxidase Substrate (Sigma)is added to all wells and incubated. After the incubation period, theplates are read at 450 nm on a microtiter plate reader.

Anti-Wise antibodies are further characterized by their reactivity withthe mouse bone, tooth, kidney, and other tissue, including but notlimited to osteoblasts and osteoclasts. Monoclonal or polyclonalanti-Wise antibodies can be tested in an immunohistological assay usingtissues, biochemically in an immunoprecipitation assay, and functionallyin a Wnt pathway activation or inhibition assay. Briefly, anti-Wiseantibodies are tested for reactivity with a panel of mouse sectioned orwhole mount tissues and by immunofluorescence staining with fluoresceinor rhodamine conjugated goat anti-mouse or rabbit immunoglobulin heavyor light chain reagents (TAGO, Burlingame, Calif.) using standardtechniques. See Thielmans, K., et al., J. Immunol. 133:495 (1984) andSamoszuk, M. K., et al, Hybridoma 6:605 (1987). Other colorimetricimmunological reagents may be utilized in this immunohistologicalmethod. Alternatively, tissue-derived cell suspensions can be analyzedby either fluorescence microscopy or flow cytometry using a fluorescenceactivated cell sorter (Becton Dickinson FAXS 440, Mountain View,Calif.).

In a biochemical functional assay, anti-Wise antibody may be used tobind Wise protein or polypeptide, thereby inhibiting binding of Wise toLRP. Wise-FLAG and LRP-MYC reagents are made such that addition ofanti-Wise antibody prevents Wise binding to LRP. In addition, anti-Wiseantibody may immunoprecipitate Wise-FLAG, forming an antibody-antigencomplex that is then detectable on Western blot analysis. Therefore,this assay may be used to detect anti-LRP antibody activity infunctional inhibition of Wise-LRP binding. This functional assay is usedas a screening tool to obtain antibodies, both monoclonal andpolyclonal, which functionally bind to Wise protein in vitro and in vivoand prevent Wise binding to LRP. Similarly, anti-LRP antibodies may bescreened. It is predicted that such therapeutic anti-Wise antibodies andanti-LRP antibodies can be used in vitro and in vivo to increaseosteoblast number and bone mineral density and bone deposition.

In a luciferase assay, anti-Wise antibody may function to activate theWnt pathway. Here, Human293 cells are used wherein anti-Wise antibodybinds to Wise and prevents such Wnt pathway inhibition.

Upon completion of testing of anti-Wise antibodies in at least one ofthe above assays, those mouse sera and rhybridoma clones producingmonoclonal antibodies that are reactive against Wise present in bonecells (osteoblasts, osteoclasts) can be selected for further expansionand processing. Goat antisera containing polyclonal antibodies reactiveagainst Wise can also be produced.

Hybridoma production can be carried out by fusing the mouse spleen cellswith the myeloma X63AG8.653 cell line by the procedure described inHarlow, E. and D. Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, 1988. The Sp-2/0 myeloma cell line may also be used.Briefly, spleen cells are mixed with HAT-sensitive X63AG8.653 myelomacells and fused with polyethylene glycol (PEG) (e.g., 50% PEG 4000,Sigma Chemicals). Subsequent to fusion of spleen cells with the myelomacell line, 1 drop of the 50 ml fusion mixture is added to each of 96wells in 10 microwell cell culture plates (Corning). After culture ofclones in selection media, hybridoma cultures are screened for antibodyproduction to Wise antigen as follows:

Wise-polypeptide coated wells are prepared. Further, BSA is coated onwells following the same coating procedure as with BSA-Wise to detectany nonspecific binding. The antigen coated plates are used to screencell culture supernates from each of the parental cultures. The parentalsupernates are added to one well of BSA-Wise-coated microtiter plate andto one well of BSA coated plate. The plates are incubated and washed.Goat anti-mouse IgG (gamma chanin specific) horseradishperoxidase-conjugated antibody is added to each well. Parental culturesare identified that produce absorbance readings exceeding 0.3 O.D. onthe BSA-Wise wells and no reactivity on the BSA coated wells. The latterparental cultures are expanded in culture in 24 well macrowell plates(Corning) and upon further supernatant/antibody evaluation, threeparental cultures are re-cloned (secondary cloning). Following aprocedure described in Harlow and Lane, supra, the parental cultures arediluted in RPMI 1640 culture medium containing 20% fetal bovine serum togive a cell density of 0.5-10 cells per well on wells that areprecultured with splenocyte feeder cells.

After two weeks parental cell culture supernates are tested to determinethe wells that are positive for monoclonal anti-Wise antibody activityusing the screening procedure above. Positive wells are cloned andsubcloned. Clonal cultures can be identified with high viability andproducing the highest titer antibody to BSA-Wise in the aforementionedantibody screening assay. Secondary and tertiary subcloning of thelatter is done to assure monoclonality and stability of the resultantclones. Comparative affinity analysis may be performed in accordancewith Macdonald et al. (Macdonald, R. A. et al. 1988. Journal ofImmunological Methods, 106:191-194). The cells from each culture areprepared in accordance with Harlow and Lane, supra, for frozen storagein ampoules in liquid nitrogen. Each single clone is expanded in cultureand adapted to a protein-free medium (MaxiCell/Hybridoma-PF Medium, Cat.No. N10105, Atlanta Biologicals, Norcross, Ga.) for monoclonal antibodyproduction. Thus, anti-Wise monoclonal antibodies are prepared that canbe utilized in subsequently described bone deposition experiments.

Next, monoclonal antibodies from subclones can be tested against Wisewild type and mutant polypeptides for binding by direct ELISA andcompetition ELISA methods. For direct ELISA, BSA-Wise is coated onmicrotiter plates, the unbound sites are blocked by incubation withAssay Buffer (25 mM borate, pH 8.0, 150 mM NaCl, 0.01% EDTA and 1% BSA).The plate is washed 6× and increasing concentrations of monoclonalantibody (mAb) in Assay Buffer are added. After this incubation, theplate is again washed and incubated with alkaline-phosphates labeledgoat anti-mouse antibodies (Cappel, Durham, N.C.) diluted 1:1000 inAssay Buffer. The unbound antibodies are removed by extensive washingand the bound antibodies are detected by addition ofp-nitrophenylphosphate (PNPP). The optical density at 410 nm isrecorded.

The competition ELISA can be performed by pre-coating microtiter plateswith BSA-Wise wild type and mutant polypeptides and blocking with AssayBuffer. The plate is washed, and monoclonal anti-Wise antibody is addedwith increasing, concentrations of the Wise wild type and mutantpolypeptide antigen competitors, simultaneously incubating the mixturefor 1 hr at 37° C. The unbound materials are removed by extensivewashing and the bound mAb is detected with alkaline phosphatase labeledanti-mouse antibodies similar to the direct ELISA method above. Allwashes are in TBS-T wash solution; all incubations proceeded for 1 hr at37° C. It is predicted that monoclonal antibodies directed against Wiseimmunogen will bind specifically to Wise wild type molecules. Suchanti-Wise monoclonal antibodies, depending on their reactivity profiles,may or may not bind to Wise mutant molecules that do not bind to LRP.

Fab fragments of anti-Wise antibodies can be prepared. Afterpurification of anti-Wise IgG antibody, Fab fragments are prepared bypapain cleavage. Mercuripapain is pre-activated with 10 mM cysteine in1.25 MM EDTA for 15 min at 37° C., then added to the IgG antibody (5-10mg/ml) at a 1:50 to 1:200 (w/w) ration of enzyme to antibody. The periodof incubation at 37° C. ranged between 15 min to 5 hours to determinethe optimum time of incubation for maximal Fab yield. Addition ofiodacetamide (20-50 mM) stopped the cleavage process. Conditions areoptimized by SDS-PAGE. analysis of resultant reaction products.

Thus, anti-Wise monoclonal antibodies and Fab “mini-antibody” fragmentsare prepared that can be utilized in subsequently described experimentsbelow wherein such antibodies are delivered in liposomes to bone cells(e.g., osteoblasts) for the purpose of increasing bone deposition andbone mineral density in vitro and in vivo. Anti-Wise Fab fragments arepredicted to have greater anti-Wise inhibitory activity than wholeanti-Wise antibody. Both anti-Wise antibodies and their correspondingFab fragments are expected to bind to Wise molecules in osteoblasts andprevent Wise molecule binding to LRP molecules (e.g., LRP5, 6).

Example 32

Hybridoma cell lines can be prepared that can secrete monoclonalantibodies reactive with Sost wild type proteins, polypeptides, wholemolecules, and fragments according to the procedure described in Example31 above. Briefly, murine myeloma cells are fused with murine spleniclymphocytes from mice immunized with Sost-derived antigen. Hybridomasmaking monoclonal antibodies reactive against Sost antigen are selected,grown, and monoclonal antibodies can then be screened with Sost antigenin EIA assays, histological tissue staining assays, immunoprecipitationassays, and functional assays as previously described. Fab fragments ofanti-Sost antibodies can be prepared by standard papain and pepsinenzymatic digestion methods.

Example 33

This Example relates to detection and analysis of the wild type, andalso genetically modified, Wise cysteine knot regions in mammaliancells. Similarly, detection and analysis of the Sost cysteine knotregion from wild type or genetically modified cells may be executed. Inthis procedure, murine C57BL/6 osteoblasts, producing Wise polypeptideare isolated. Other isolated or cultured mammalian cells can be used.Genetically modified Wise molecules can be made as presented in Example18-21, wherein the stop codon in the Wise Neo-lacZ cassette, which issubsequently inserted into ES cells, encodes a truncated Exon 2polypeptide product that comprises part of the cysteine knot region ofWise, After PCR amplification of these shortened Wise nucleic acidsequences by standard molecular biology cloning techniques, suchsequences are placed on Southern blots for gDNA and on Northern blotsfor mRNA species. J. Sambrook and D. W. Russell, Molecular Cloning: ALaboratory Manual, 3^(rd) edition (2001).

More specifically, Wise gene nucleic acid fragment sequences for SEQ IDNOs 1-5 and 126-128 may be made and amplified by standard PCRtechnologies. These Wise nucleic acid sequences encode correspondingpolypeptides. A smaller Wise gene DNA or RNA probe sequencecorresponding to SEQ ID NO 1 can be synthesized (see SEQ ID Nos136-140). Alternatively, site specific mutagenesis or in vitrotranscription methods may be utilized. The DNA probe can then be labeledwith P-32 cytosine (CTP). Alternatively, C-14, H-3, or other radiolabelsor nonradioactive labels (e.g., DIG) may be used. In addition, the RNAprobe can be labeled with P-32 uracil. Once Wise DNA probes are labeledwith P-32 cytosine, these radiolabeled probes may be hybridized tonucleic acids extracted from Wise-containing cells to characterize suchWise genes after Southern blot analysis. Similarly, radiolabeled WiseRNA probes may be hybridized to nucleic acids from Wise-containing cellextracts. It was observed that these Wise RNA fragments detected thepresence of Wise nucleic acid sequences in the cell extracts.

Example 34

Wise antigens to be prepared for immunization and to be used asstandards in immunoassays include, but are not limited to, Wise wildtype polypeptide whole molecule and polypeptide fragments. In addition,the corresponding Wise-derived nucleic acid molecules to theaforementioned polypeptide molecules were produced as antigens forimmunizations and standards. Both Wise-derived polypeptide and nucleicacid antigens are prepared as previously described herein.

Goat and rabbit polyclonal antibodies and mouse monoclonal antibodies tothe Wise-derived wild type and mutant polypeptide and nucleic acidmolecules are prepared by methods that are known to those of skill inthe art. E. Harlow and D. Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1988. Similarly, goat and rabbitpolyclonal antibodies and mouse monoclonal antibodies may be made toSost-derived wild type and mutant polypeptides and nucleic acidmolecules. The procedure for production of monoclonal antibodies tospecific antigens has been described in detail herein. Once monoclonaland polyclonal antibodies to Wise-derived polypeptide and nucleic acidmolecules have been made, they can be utilized in immunodiagnostics kitassays for the detection and quantitation of the Wise-derived molecules.

Example 35

A Sost-specific monoclonal antibody can be made by the procedure asdelineated in Examples 32 and 33. Sost-specific monoclonal antibodiesmay be made against Sost wild type and mutant proteins and polypeptides.These antibodies would inhibit the binding of Sost to LRPs.

Example 36

This Example relates to the production of monoclonal antibody to theterminus region of LRP5 which binds to Wise protein. This LRP5 terminusregion also binds to Sost protein. The anti-LRP5 antibody is predictedto inhibit binding of Wise to LRP5 and thereby result in phenotypicchanges such as increased osteoblast number, increased bone mineraldensity and bone deposition, and tooth and ocular phenotypic changes.Similarly, the anti-LRP5 antibody is predicted to inhibit binding ofSost to LRP5, resulting in similar phenotypic changes.

LRP 5 Δ3-4 mutants are made as described in Examples 28 and 29. SuchLRP5 Δ3-4 mutant nucleic acid sequences can be inserted into either p28bvectors as described in Examples 28 and 29, or Neo-lacZ cassettes(without stop codons) as described in Example 13. E. coli cellscontaining the p28b vector with the LRP5 mutation, and ES cellscontaining the Neo-lacZ cassette with the same mutation are cultured,lysed, and LRP5 Δ3-4 purified.

Monoclonal antibody can be made to LRP5 by immunization of mice withLRP5 as described in Example 32, hybridization of LRP5 immunized mousesplenic lymphocytes with HAT-sensitive myeloma cells, and selection ofHAT-resistant hybridoma cells secreting antibodies that bind LRP5. Onceclones are identified that secrete antibody binding to LRP5, clones arefurther screened for failure to bind LRP5 Δ3-4 in EIA and functionalassays as described in Example 32. Such hybridoma clones that bind towild type LRP5 molecules yet do not bind to LRP5 Δ3-4 molecules aredeemed to be putative anti-LRP5 Δ3-4 region-epitope specific antibodies(LRP5 Δ3-4). Photoreactive chemical conjugation of H3-radiolabeledantibody combining sites to the LRP5 molecule can verify thisantibody-specific attachment to the terminal amino acid sequence ofLRP5.

Example 37

This Example relates to the production of monoclonal antibody to theterminus region of LRP6 which binds to Wise protein. The anti-LRP6antibody is predicted to inhibit binding of Wise to LRP6 and therebyresult in phenotypic changes such as increased osteoblast number,increased bone mineral density and bone deposition, and tooth and ocularphenotypic changes. Similarly, the anti-LRP6 antibody is predicted toinhibit binding of Sost to LRP6, resulting in similar phenotypicchanges.

LRP 6 M-4 mutants are made as described in Examples 28-29. Such LRP 6Δ3-4 mutant nucleic acid sequences can be inserted into either p28bvectors as described in Examples 28-29, or Neo-lacZ cassettes (withoutstop codons) as described in Examples 13. E. coli cells containing thep28b vector with the LRP6 mutation, and ES cells containing the Neo-lacZcassette with the same mutation are cultured, lysed, and LRP6 Δ3-4purified.

Monoclonal antibody can be made to LRP6 by immunization of mice withLRP6 as described in Example 32, hybridization of LRP6 immunized mousesplenic lymphocytes with HAT-sensitive myeloma cells, and selection ofHAT-resistant hybridoma cells secreting antibodies that bind LRP6. Onceclones are identified that secrete antibody binding to LRP6, clones arefurther screened for failure to bind LRP 6 Δ3-4 in EIA and functionalassays as described in Example 32. Such hybridoma clones that bind towild type LRP6 molecules yet do not bind to LRP 6 Δ3-4 molecules aredeemed to be putative anti-LRP6 Δ3-4 region-epitope specific antibodies(LRP6 Δ3-4). Photoreactive chemical conjugation of H3-radiolabeledantibody combining sites to the LRP6 molecule can verify thisantibody-specific attachment to the terminal amino acid sequence ofLRP6.

Example 38

This Example relates to the production of biotinylated liposomes whichare then linked to monoclonal antibodies specific for osteoblaststhrough an avidin linkage. These anti-osteoblast antibody-armedliposomes can be utilized to deliver encapsulated anti-Wise antibody toosteoblasts. Similarly, encapsulated anti-Sost antibody may be made anddelivered to osteoblasts. Liposomes may be armed with anti-osteoblast(anti-OB) antibodies that react with either mouse or human osteoblastsas described herein. Delivery of anti-Wise antibodies to osteoblastsusing encapsulated liposomes is anticipated to result in increasedosteoblast growth and proliferation with concommitant increased bonedeposition.

Biotinylated phospholipids are initially prepared. Biotinylatedphospholipids are prepared by dissolving phosphatidylethanolamine (PE,5.1 mg) or phosphatidylserine (PS, 3.9 mg) in a solution (170 μl for PE;130 μl for PS) of chloroform-methanol (2:1) with biotinylN-hydroxysuccinimide ester (BNHS, 3.3. mg) (Sigma Chemicals, St. Louis,Mo.). 10 μl is added of a chloroform solution containing 15% (v/v)triethylamine. After a two hour incubation of the reaction mixture atambient room temperature (18° C.), the crude mixture is stored at −70°C.

The crude biotinylated lipid is then purified by high-performance liquidchromatography (HPLC) using a Waters system (Waters Associates, Milford,Mass.) with two solvent delivery units (M-45 and Model 510) and a Model680 gradient controller. Separations are performed using a stainlesssteel column (250×4.6 mm i.d.) packed with 5 μm Lichrosorb Si-100 silica(Merck, Darmstadt, Germany) at room temperature with a flow rate of 1ml/min. After a first wash with solvent A (n-hexane/2-propanol/sater ina ratio of 60:80:14, v/v/v), Solvent B (n-hexane/2-propanol/water60:80:7, v/v/v) is added until a new baseline is stabilized.

10 μl of the crude biotinylated lipid starting reaction mixturecontaining 390 nmol lipid is applied to the HPLC column using a Hamiltonsyringe, and the elution is monitored utilizing an M-441 UV detector(214 nm). The column is eluted for 5 min with solvent A, then with a 20min linear gradient between 0 and 100% solvent B in A. Solvent B is thenpassed over the column until a stable baseline is obtained.

The average retention times of BPE and BPS are 20.7 min (17-22) and 27.1min (26-28), respectively. The HPLC peaks are collected in a GilsonMicrofractionator, and the eluted material is pooled. The solvent isthen evaporated under a stream of nitrogen, and the biotinylated lipidis stored at −70° C.

Both the initial crude reaction mixture and the HPLC-purified BPE andBPS fractions are analyzed by thin-layer chromatography (TLC) in silicagel-coated plates (Riedel-de Haen, Germany). For BPE plates, achloroform/methanol/water (80:25:2) solution is used; and for BPSplates, a chloroform/methanol/acetic acid (30:4:3) solution is used.Phospholipid visualization occurs through one of three methods: (1)exposure to iodine vapors, (2) a biotin-specific spray(dimethylaminocinnamaldehyde) See D. B. McCormick and J. A. Roth Methodsin Enzymology 148A: 383 (1987), or (3) a phosphate-specific spray. SeeV. E. Vaskovsky and E. Y. Kostetsky, J. Lipid Res. 9: 396 (1968). Allthree staining methods reveal that BPE has an R_(f)=0.65, and BPS has anR_(f)=0.55.

Biotinylated liposomes are then prepared. Biotinylated phospholipids(BPE or BPS) are dissolved in chloroform/methanol (2:1) and molarequivalents of each corresponding lipid (BPE or BPS) are added to 12mm×75 mm glass tubes to yield the final percentage of biotinylated lipiddesired (e.g., 5, 10, 20%). Concentrations of 0.01 to 1 mol % of totallipid are achieved.

To prepare liposomes, the biotinylated lipid/native lipid mixture (e.g.,2 μmol of the stock lipid mixture in chloroform/methanol) is evaporatedto dryness under a stream of nitrogen and then placed in a vacuumdessicator overnight. The lipid is resuspended by syringe injection(e.g., 50 μl lipid in chloroform/methanol into 1.0 ml PBS) in a finalconcentration of 1 mg/ml in PBS, pH 7.2-7.4, then sonicated undernitrogen in an ice-cooled chamber for 10 min in a Branson Sonifier Model130. The resulting suspension is centrifuged at 10,000 rpm for 20 min,and the biotinylated liposomes in the supernatant fraction used within24 hours after preparation.

To encapsulate anti-Wise antibodies, the biotinylated lipid/nativemixture is resuspended by injection (e.g., 50 μl lipid inchloroform/methanol into 1.0 ml PBS) into an anti-Wiseantibody-containing PBS solution. After sonication and centrifugation at10,000 rpm for 20 minutes, anti-Wise antibody-biotinylated liposomes arepurified by one of the following procedures: (1) in one preferredprocedure, liposome preparations are centrifuged at 13,000×g in amicrocentrifuge; pelleted liposomes are washed with PBS, and pelletedliposome fractions are resuspended in PBS buffer for use; (2) in anothermethod, liposome preparations are passed over a Sephadex 200 column(Pharmacia, Piscataway, N.J.) in PBS. The liposomes are eluted in thePBS void volume, with free protein and contaminants appearing insubsequent collection.

Once the liposomes are prepared, Fab fragments of anti-osteoblast cell(anti-OB) antibodies must be prepared. Rat anti-mouse Thy-1 monoclonalantibody and mouse anti-human Thy-1 monoclonal antibody are obtainedfrom Pharmingen (San Diego, Calif.). Thy 1 is known to be an expressedsurface antigen on osteoblast cells. X-D Chen, et al., Thy-1 AntigenExpression by Cells in the Osteoblast Lineage, J. Bone & MineralResearch 14(3): 362-375 (1999). Other suitable osteoblast-reactiveantibodies have been described. See, e.g., Aubin, J. E. et al.Monoclonal antibodies as Tools for Studying the Osteoblast Lineage,Microsc Res Tech 33:128-140; Bruder S P et al. (1996) MonoclonalAntibodies Selective for Human Osteogenic Cell Surface Antigens, Bone21:225-235. After purification of anti-OB IgG antibody, Fab fragmentsare prepared by papain cleavage. Mercuripapain is pre-activated with 10mM cysteine in 1.25 mM EDTA for 15 minutes at 37° C., then added to theIgG antibody (5-10 mg/ml) at a 1:50 to 1:200 w/w) ratio of enzyme toantibody. The period of incubation at 37° C. ranged between 15 minutesto 5 hours to determine the optimum time of incubation for maximal Fabyield. Addition of iodoacetamide (20-50 mM) stopped the cleavageprocess. Conditions are optimized by SDS-PAGE analysis of resultantreaction products.

Biotinylated Fab fragments of anti-OB antibodies are obtained by usingan N-hydroxysuccinimidobiotin (NHS-Biotin, Sigma Chemical, St. Louis,Mo.). In this method, 2 mg of Fab fragments are dissolved in 1 ml ofsodium phosphate buffer (PBS), pH 7.5-8.5, in a 16×125 mm test tube.Immediately before use, 1 mg of NHS-Biotin is dissolved in 1 mldimethylformamide (DMF). 75 μl of the dissolved NHS-Biotin is added tothe Fab containing test tube. The tube is incubated on ice (4° C.) for 2hours. The unreacted biotin may be removed by dialysis (e.g.,Slide-A-Lyzer Dialysis Cassette) or with a D-Salting Column (PierceChemical, Rockford, Ill.). Alternative, unreacted biotin may be removedby centrifugation of the product at 1000×g for 15-30 minutes using amicroconcentrator. After centrifugation, the sample is diluted in 0.1 Msodium phosphate, pH 7.0. The process can then be repeated twice more.The biotinylated protein may be stored at 4° C. in 0.05% sodium azideprior to use.

Finally, Fab-anti-Wise liposomes utilizing biotinylated Fab molecules,biotinylated liposomes and avidin are prepared. The biotinylated Fabfragments in PBS are mixed with a twenty-fold molar excess of egg whiteavidin (Vector labs, Burlingame, Calif.; Sigma Chemical, St. Louis,Mo.), incubated overnight at 4° C. The excess avidin is removed bypassage of the mixture over anti-human light chain affinity columns(e.g., Pharmacia Sepharose 4B). Fab-biotin-avidin molecules are elutedwith citrate buffer, pH-4.0, then pooled fractions are dialyzed againstPBS, pH=7.0. A suspension of biotinylated anti-Wise antibody-containingliposomes is mixed with Fab-biotin-avidin solutions in PBS to yieldavidin to free biotin ratios on the liposome surfaces of approximately2:1, 5:1, 10:1, and 20:1 molar ratios. After incubation overnight at 4°C. on a rotational shaker, liposomes are passed through a PharmaciaSephadex G-200 column. The Fab-anti-Wise liposomes are collected in thevoid volume and resuspended in PBS.

Alternatively, biotinylated anti-Wise antibody-containing liposomes aremixed with a twenty-fold molar excess of strepafidin, incubatedovernight at 4° C., then biotinylated-avidin liposomes are passed overthe anti-human light chain affinity column. Biotinylated-avidinanti-Wise liposomes are eluted with a citrate buffer, pH=4.0, thenpooled fractions are dialyzed against PBS, pH=7.0. After dialysis,biotinylated-avidin liposomes are mixed with biotinylated Fab fragmentsin PBS in the above approximate molar ratios. Similarly, afterincubation overnight at 4° C. on a rotational shaker, liposomes arepassed through a Pharmacia Sephadex G-200 column. The Fab-Anti-Wiseantibody liposomes are collected in the void volume and resuspended inPBS.

Example 39

The present Example relates to in vitro treatment of mouse bone cells(e.g., osteoblasts and osteoclasts) by anti-Wise-specific Fab liposomesarmed with anti-osteoblast antibody as prepared in Example 40. Theanti-Wise antibody may have specificity for Wise whole molecule orpolypeptides. Similarly, anti-Sost antibody may be encapsulated inliposomes to obtain osteoblast phenotypic effects in vitro. First,murine bone cells are purified by fluorescence activated cell sorting(FACS) using anti-bone marker antibodies. In addition, mouse bone cellosteoblasts can be prepared essentially as described by Takahashi etal., 1988 Endocrinology 123:2600-2602; and by Tanaka et al., 1992 J.Bone Min Res. 7:S307, which are incorporated by reference herein. Thesebone cells are liquid nitrogen-cryopreserved in ampoules.

Bone cells are separated by fluorescence activated cell sorting (FACS)utilizing the FACStar-PLUS flow cytometer (Becton Dickinson) equippedwith two 5-watt argon ion lasers and a tunable dye laser interfaced witha Digital Equipment Corporation Vas Station-4000/90 computer and datacollection/analysis software. Bone cells are prepared by suspension inMACS (magnetic sorting) buffer with fluorescein-conjugated antibodydirected against mouse osteoblasts (DAKO, Carpenteria, Calif.) in a tubeand vortexed. After incubation for 30 minutes at 4° C., cells are washed3-5 times in MACS buffer, then centrifuged at 400×g for 5 minutes at 4°C. Cells are placed in MACS buffer at 4° C. for separation in theFACStar flow cytometer. Mouse bone cells are separated on the basis ofthe fluorescence and size. Aliquots of purified murine osteoblasts andosteoclasts cells are tested for reactivity with anti-Wise antibodies ina fluorescence sandwich immunoassay with murine monoclonal anti-Wiseantibody made according to the method of Example 32 andfluorescein-conjugated goat anti-mouse IgG antibody (H & L) (DAKO,Carpenteria, Calif.). Cells are stained for viability (>90%) by Trypanblue staining.

Subsequent to viable bone cell isolation above, bone cells (primarilyosteoblasts or osteoclasts) are incubated in vitro with anti-Wiseantibody-containing liposomes. An aliquot of anti-Wise-liposome-bonecells are then lysed. Polypeptide molecules of the elysate are separatedand characterized by SDS gel electrophoresis and Western blot analysis.Reduction of Wise binding to LRP in the presence of anti-Wise antibodyin bone cells can be measured.

It is predicted that anti-Wise antibody and anti-Wise Fab fragmentmolecules will both inhibit binding of wild type Wise to LRP5 inosteoblasts in vitro. In contrast, anti-Wise antibodies and fragmentsshould not bind to osteoclasts, nor assert an effect on osteoclastactivity (e.g., bone resorption). Correspondingly, based in part uponresults described in Example 16 herein, it is expected that anti-Wiseinhibition of Wise binding to LRP5 will result in increased growth andnumber of osteoblasts. Such increase in osteoblast number has previouslybeen associated with concomitant increases in bone deposition and bonemineral density in vivo, as described in Example 16. Thus, treatmentwith anti-Wise liposomes is predicted to result in increased bonedeposition and bone mineral density in in vivo mouse studies.

Example 40

The present Example relates to in vivo treatment of nude mice implantedwith murine bone cells (e.g., osteoblasts, osteoclasts) with anti-Wiseantibody-containing liposomes. Congenitally athymic nude mice containingmurine bone fragment implants can be used as a test system for assessingthe anti-Wise antibody-containing liposomes on murine bone cell growthin vivo according to the procedure described herein.

Congenitally athymic homozygous CD-1 female nude scid/scid mice (SCID,Charles River Laboratories, Wilmington, Mass.) are housed in sterilecages, treated with antibiotics and give autoclaved food and water. Atapproximately 6-8 weeks old, SCID mice can be injected subcutaneouslywith cut fragments of femurs and tibias of allotypically differentmurine fetuses or immature pups. Balb/c mice can be used as bone donors.Intraperitoneal injection of mice with bone fragment marrow implants isan alternative rout of administration. These mice may now be referred toas “SCID-bone mice.” Murine implanted bone fragment marrow grafts areallowed to “take” for approximately 6-8 weeks prior to injection withanti-Wise antibodies. Fetal donor bone cell suspensions are analyzed formurine allotypic markers.

As previously described herein, Fab anti-Wise antibody-containingliposomes can be prepared. 0.5-5.0×10⁶ bone osteoblast cells aresuspended in 20 ml of complete RPMI-1640 medium and injected with aHamilton microliter syringe into each of the 6-8 week old murine bonemarrow grafts of the SCID-bone mice. In the first experiment, Fabanti-Wise antibody liposomes (200:1; 100:1, 50:1 liposome:cell ratios)can be mixed together with bone cells prior to injection in vivo. In thesecond experiment, bone cells can be injected into the bone fragmentmarrow grafts, then anti-Wise antibody-containing anti-osteoblastantibody-armed liposomes (200:1; 100:1; 50:1 liposome:cell ratios) canbe injected by several routes: (1) directly into the murine bone marrow,4, 6, and 24 hr after murine bone fragment implantation; and (2)intravenously in the mouse tail vein at 0, 4, 6, and 25 hr after murinefragment implantation. Alternatively, and perhaps preferably, anti-Wiseantibody-liposomes may be mixed with osteoblasts or osteoclasts prior toplacement in operably contact with the implanted bone fragment. Controlswould include anti-Wise antibody-containing liposomes wherein theattached arming antibody lacks osteoblast-binding specificity, andliposomes lacking anti-Wise antibody.

The effect of anti-Wise antibody-liposome treatment on bone cells canthen be assessed by the following procedure. Growth of bone cells (i.e.,osteoblasts, osteoclasts) can be analyzed by examining cells harvestedfrom SCID-bone mouse bone fragment marrow implants at 1, 2, 4, 8, 16,and 32 weeks after anti-Wise antibody-liposome injection. Harvestedcells can be analyzed by flow cytometry in the FACScan system aftersuspension in complete RPMI-1640 medium, washing in RPMI, lysing of redblood cells with ammonium chloride, and staining with immunofluorescentreagents. Immunofluorescence sandwich markers including fluorescein- orrhodamine-conjugated goat anti-mouse IgG (H & L) antibody can be used inconjunction with murine monoclonal anti-Wise anti-body. Histologicalsections of bone, bone marrow, spleen, lymph node, lung and other tissuecan also be prepared 1, 2, 3, and 4 months after bone cell implantation,sectioned, and stained with immunofluorescence reagents described aboveor with hematoxylin and cosin-stained formalin-fixed andparaffin-embedded specimens compared with specimens from untreated,control SCID-bone mice in which no osteoblast or osteoclast cells areinjected. Significantly, SCID-bone mice may be analyzed for treatmentwith anti-Wise antibody-liposomes to determine efficacy of suchliposomes to increase in growth and number of osteoblasts which isexpected to result in increased bone deposition in this in vivo SCIDnude mouse model system. Moreover, SCID-bone mice may be used to assessanti-human Wise antibody treatment effects utilizing xenogeneic humanbone fragment transfers into such SCID-bone mice as described herein.

Example 41

This Example relates to injection of anti-Wise antibodies into the pupsof the C57BL/6 mouse strain to determine their positive effects onWise-regulated phenotypes. Alternatively, the 129 mouse strain may beused. Monoclonal and polyclonal anti-Wise antibodies specific for wildtype Wise polypeptide molecules were made as described in Example 31above. The antibody can be directed to the cysteine knot-containingregion of Wise. Similarly, anti-Sost antibodies may be injected intomouse pups to determine phenotypic and therapeutic changes.

In this procedure, C57BL/6 mouse pups are injected with therapeuticdoses of anti-Wise antibody in a pharmaceutical carrier such as sterileendotoxin-free phosphate buffered saline (PBS) at 2, 4, 6, 8, 10, 12,14, 16, 18, and 20 days post partum. Alternatively, anti-Wise antibodyFab fragments may be used in a suitable pharmaceutical carrier medium.Bone mineral density of injected mice is compared to that of uninjectedcontrol mice as described in Example 16. It is anticipated thatanti-Wise antibody treatment will result in increased bone mineraldensity and increased bone deposition in injected mice as compared tocontrols.

It is also predicted that anti-Wise antibody treatment will result inphenotypic changes in eyes and teeth as described in Wise mutant mice inExamples 15 and 17 above. Thus, anti-Wise antibody injected mice areexpected to exhibit loss of optic nerve fibers and increased rod andcone layers in the retina as shown in Example 15 in Wise mutant mice.Anti-Wise antibody injected mice are predicted to manifest molar andincisor tooth abnormalities similar to those of Wise mutant mice asdemonstrated in Example 17. An additional incisor tooth phenotype notpresent in the wild type mouse may be observed. In addition, anti-Wiseantibody injected mice may show an additional M1 molar tooth, with anadditional associated root. Anti-Wise antibody injected mice may alsoexhibit reverse orientation patterning of molar teeth, with possiblefusion of M1 and M2 teeth.

Example 42

In this Example, kit components for detection and quantitation of Wisewild type and mutant polypeptides and fragments are described.Immunodiagnostics methodologies utilized in these kits are modificationsof general and specific principles well known in the art. E. Harlow andD. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, and E. T. Maggio, Ed., Enzyme-Immunoassay, CRC Press,Florida, 1980 are incorporated by reference herein.

Sandwich enzyme immunoassay kit components are as follows: 96-wellmicrotiter plates coated with anti-Wise antibody, diluent buffer, Wisestandards, horseradish peroxidase (HRP)-conjugated mouse anti-Wiseantibody, ortho-phenylenediamine (OPD) substrate solution, containingH₂O₂, and 2N sulfuric acid stop solution.

Competitive enzyme immunoassay kit components are as follows: 96-wellmicrotiter plates coated with wild type or variant Wise molecules,diluent buffer, Wise wild type and variant standards, horseradishperoxidase (HRP)-conjugated mouse anti-Wise antibody,ortho-phenylenediamine (OPD) substrate solution, containing H₂O₂, and 2Nsulfuric acid stop solution. Similarly, Sost immunoassay kits may beprepared by substituting anti-Sost antibody for anti-Wise antibody, andSost standards for Wise standards as components in the above describedWise kit.

Example 43

In this Example, an immunoprecipitation protocol and subsequent WesternBlot protocol are described for analysis and characterization of variousWise-derived proteins and polypeptide molecules. Similarly,immunoprecipitation and Western blot analysis and characterization maybe executed for Sost-derived proteins and polypeptide molecules. Westernblot kits based on the methodology described herein may also beproduced.

Western blot kits will contain the following components: Wise-derivedprotein and polypeptide molecule standards, primary goat antibodyagainst Wise, secondary alkaline phosphatase-conjugated anti-goatantibody, blocking buffer, diluent buffer, and substrate developmentsolution.

The immunoprecipitation protocol involves a technique for separation ofWise-derived polypeptide molecules from whole cell lysates or cellculture supernatants. Wise-derived polypeptide molecules may be wildtype or mutant molecules; and these molecules may be obtained frommammalian cell cultures (e.g., osteoblasts) or from bacterial cells(e.g., E. coli) or mammalian cells. After immunoprecipitation binding toanti-Wise antibody and separation of these Wise-derived polypeptidemolecules, the Wise molecules can be identified, biochemicallycharacterized, and expression levels quantitated. Sost-derivedpolypeptide molecules may be similarly immunoprecipitated.

In initial immunoprecipitation runs, approximately 5-10 μg ofanti-Wise-derived polypeptide molecule antibody is added to an Eppendorftube containing the cold precleared lysate containing Wise polypeptides.Alternatively, antibodies recognizing the MYC tag may be utilized forthese immunoprecipitations of Wise polypeptides. Reduced and nonreducedWise-derived polypeptide molecules are prepared to run alongsideprestained molecular weight standards for use on SDS-PAGE gels.

In the R&D System Immunostaining procedure, Western Blot membranes areblocked in Blocking Buffer, incubated with primary goat anti-Wisepolypeptide antibody, incubated with secondary antibody (e.g., alkalinephosphatase conjugated anti-goat IgG antibody), incubated with SubstrateDevelopment solution, dried, and blocked in Blocking Buffer. Blockunoccupied protein binding sites on membrane by placing membrane inBlocking Buffer on a rocker/shaker. Primary antibody (e.g., goatanti-Wise polypeptide molecule antibody) in Diluent Buffer is added tothe membrane and incubated. After washing, incubate blots with 20 mL ofsecondary antibody (e.g., TAGO alkaline phosphatase-conjugated rabbitanti-goat IgG antibody) in Diluent Buffer and incubated. Wash membranes,incubate and then add Substrate Development Solution to membrane. Stopsubstrate development after incubation by pouring off DevelopmentSolution and rinsing membrane in deionized water.

In summary, this Western blot methodology can be used to identify,biochemically and immunologically characterize, and quantitate Wise andSost polypeptide molecules derived from wild type and/or mutants in bothmammalian and bacterial cell culture systems. In addition, Western blotkits may be produced utilizing Wise-derived and Sost-derived moleculestandards, antibodies, and kit components described and utilized in theabove-described methodology.

Example 44

In this Example, hybridization kits are described for the detection ofWise wild type and Wise variant nucleic acid sequences. Wise wild typeand variant nucleic acid sequence molecules are prepared by either PCRmethodology [Mullis, U.S. Pat. No. 4,683,195; Mullis, 4,683,202],including real time PCR techniques, or conventional cloning technologyas described in Examples 19-20. Probe nucleic acid sequences can beproduced in vectors as described previously. As alternatives to PCRmethodology, isothermal techniques [Guatelli et al., Proceeding of theNational Academy of Science 87: 1874-1878 (1990)], transcription basedmethods [Kwoh et al., Proceedings National Academy of Science 86:1173-1177 (1989)], and QB replicase techniques [Munishkin et al., Nature33: 473 (1988)] may be used. DNA or RNA primers are prepared containingdesired Wise or Sost probe sequences. For example, a nucleic acid probecan be prepared to different portions of Wise nucleic acid sequences.Similarly, probes can be prepared for nucleic acid sequences that encodeinactive Wise polypeptide variants that either do not bind to LRP5 orLRP6 or, alternatively, that, when inserted into mammalian cells, causephenotypic increases in bone deposition or bone mineral density. [Kempet al., Proceedings of the National Academy of Science 86: 2423-2427(1989)].

Wise wild type molecule and Wise variant cDNA synthesis and DIG labelingis as follows: Heat 10-15 μg Wise sample RNA with 1.7 μl random primers(3 ug/ul; Invitrogen Cat. No. 48190-011) and 15.9 μl H₂O at 70° C. Snapcool on ice and centrifuge. To each reaction tube, add DIG-dCTP. AddMaster mix as follows: First Strand Buffer, DTT, dNTPs (25 mM eachdA/G/TTP, 10 mM dCTP), SuperScript II (200 U/ul; Invitrogen Cat. No.18064-014). Incubate reaction at 25° C., followed by 42° C. incubation.

While incubating the above reaction mixture, slides are prepared forhybridization as follows: Incubate the prehybridization solution in aCoplin jar at 63° C. to equilibrate. Place slides in the pre-heatedsolution and incubate at 63° C. Prepare two staining troughs, one withMilliQ H₂O and the other with isopropanol. Place slides in slide rackand immerse in first trough to rinse in MilliQ H₂O with vigorousshaking. Transfer the rack into the second trough and rinse inisopropanol. Dry slides by centrifugation on a microtiter plate rotor onabsorbent cloth. Store slides in slide box prior to hybridization.

Briefly centrifuge the labeling reaction tubes. Add 10 μl 1N NaOH andheat at 70° C. to hydrolyze the RNA. Neutralize by adding 1 N HC 1.Using the MinElute PCR purification kit (Qiagen Cat. No. 28004), combineDIG-labeled cDNA samples in a single Eppendorf tube and add Buffer PB.Apply to MinElute column in collection tube and centrifuge. Purplecoloration of the membrane indicates efficient labeling of both cDNAsamples. Add 50 μl Buffer PE to MinElute column and centrifuge to drythe membrane. Add 10 μl MilliQ H₂O pH 7-8.5 carefully to the center ofthe membrane and allow to stand for 1 min. Centrifuge to collect cDNA(yield 80%). Place the MinElute column into a fresh tube B. Add MilliQH₂O pH 7-8.5 to the center of the membrane and allow to stand for 1 min.Centrifuge at 13,000 rpm for 1 min to collect residual cDNA. Transfer4.5 μl from tube B to tube A (final volume 14.5 μl).

For hybridization, the following procedure is used: Mix purified DIGsample with hybridization solution (DIG-labeled cDNA, filtered 20×SSC,filtered 2×SDS). Prepare a slide heating block. Preheat thehybridization chamber. Heat hybridization solution at 99° C. for 2 minto denature cDNA. In the meantime, prepare the slide and a 24×24 mmcoverslip. When ready, immediately centrifuge the hybridization solutionbriefly, put the slide into the chamber, pipet SSC into each of the twowells of the chamber, and apply the solution onto the slide at the edgeof the spotted area avoiding bubble formation by using curved-edge fineforceps to set the coverslip in place. Close the chamber and immerse itin a 63° C. waterbath. Incubate chambers overnight.

Transfer slides one at a time from the chamber to the Coplin jarcontaining Wash A and let the coverslip fall off by gently moving theslide vertically in the solution. Once the coverslip is removed,transfer the slide quickly to the rack in the trough of Wash A. When allslides are on the rack, wash by vigorous agitation for 5 min at roomtemperature. Transfer the slides quickly to the rack in the secondtrough containing Wash B. Wash by vigorous agitation for 3 min at roomtemperature. Transfer the rack to the third trough containing Wash B andwash by vigorous agitation for 3 min at room temperature. Dry slides andstore in a slide box until scanning.

The ScanArray Express (Perkin Elmer Life Sciences, Boston, Mass.) can beused to scan the slides. Alternatively, the Image Trak Eip-FluorescenceSystem (Perkin Elmer Life Sciences, Boston, Mass.) can be used for96,384, or 1536 well plates.

In summary, hybridization methodology and kits for the detection,identification, and quantification of Wise-associated nucleic acidsequences in cells are set forth herein. Using these methods, Wise wildtype and mutant nucleic acid sequences can be identified, characterized,and quantified. In addition, kits may be produced utilizing Wise-derivednucleic acid molecule standards, antibodies, and kit components asdescribed in the above methodology.

REFERENCE LIST

-   Amaya, E., Musci, T. J., and Kirschner, M. W. (1991). Expression of    a dominant negative mutant of the FGF receptor disrupts mesoderm    formation in Xenopus embryos. Cell 66, 257-70.-   Axelrod, J. D., Miller, J. R., Shulman, J. M., Moon, R. T., and    Perrimon, N. (1998). Differential recruitment of Dishevelled    provides signaling specificity in the planar cell polarity and    Wingless signaling pathways. Genes Dev. 12, 2610-2622.-   Baker, J. C., Beddington, R. S., and Harland, R. M. (1999). Wnt    signaling in Xenopus embryos inhibits bmp4 expression and activates    neural development. Genes Dev 13, 3149-59.-   Beddington, R., and Robertson, E. (1998). Anterior patterning in    mouse. Trends in genetics 14, 277-284.-   Beddington, R., and Robertson, E. (1999). Axis development and early    asymmetry in mammals. Cell 96, 195-209.-   Blumberg, B., Bolado, J., Moreno, T., Kintner, C., Evans, R., and    Papalopulu, N. (1997). An essential role for retinoid signaling in    anteroposterior neural patterning. Development 124, 373-379.-   Bork, P. (1993). The modular architecture of a new family of growth    regulators related to connective tissue growth factor. FEBS 327,    125-130.-   Bourguignon, C., Li, J., and Papalopulu, N. (1998). XBF-1, a winged    helix transcription factor and dual activity, has a role in    positioning neurogenesis in Xenopus competent ectoderm. Development    125, 4889-900.-   Bradley, L., Sun, B., Collins-Racie, L., LaVallie, E., McCoy, J.,    and Sive, H. (2000). Different activities of the frizzled-related    proteins frzb2 and sizzled2 during Xenopus anteroposterior    patterning. Dev Biol 227, 118-32.-   Brannon, M., Gomperts, M., Sumoy, L., Moon, R. T., and Kimelman, D.    (1997). A β-catenin/XTcf-3 complex binds to the siamois promoter to    regulate dorsal axis specification in Xenopus. Genes Dev. 11,    2359-2370.-   Cadigan, K. M., and Nusse, R. (1997). Wnt signaling: a common theme    in animal development. Genes Dev 11, 3286-305.-   Capdevila, J., Tabin, C., and Johnson, R. L. (1998). Control of    dorsoventral somite patterning by Wnt-1 and 3-catenin. Dev Biol 193,    182-94.-   Christian, J. L., and Moon, R. T. (1993). Interactions between    XWnt-8 and Spemann organizer signaling pathways generate    dorsoventral pattern in the embryonic mesoderm of Xenopus. Genes    Dev. 7, 13-28.-   Condie, B. G., Brivanlou, A. H., and Harland, R. M. (1990). Most of    the homeobox-containing Xhox 36 transcripts in early Xenopus embryos    cannot encode a homeodomain protein. Mol. Cell. Biol. 10, 3376-3385.-   Cox, W. G., and Hemmati-Brivanlou, A. (1995). Caudalization of    neural fate by tissue recombination and bFGF. Development 121,    4349-4358.-   Danielian, P. S., and McMahon, A. P. (1996). Engrailed-1 as a target    of the Wnt-1 signalling pathway in vertebrate midbrain development.    Nature 383, 332-4.-   Dickinson, M. E., Selleck, M. A., McMahon, A. P., and    Bronner-Fraser, M. (1995). Dorsalization of the neural tube by the    non-neural ectoderm. Development 121, 2099-106.-   Doniach, T. (1993). Planar and vertical induction of anteroposterior    pattern during the development of the amphibian central nervous    system. J. Neurobiology 24, 1256-1275.-   Ensini, M., Tsuchida, T., Belting, H.-G., and Jessell, T. (1998).    The control of rostrocaudal pattern in the developing spinal cord:    Specification of motor neuron subtype identity is initiated by    signals from paraxial mesoderm. Development 125, 969-982.-   Fagotto, F., Guger, K., and Gumbiner, B. M. (1997). Induction of the    primary dorsalizing center in Xenopus by the Wnt/GSK/∃-catenin    signaling pathway, but not by Vg1, Activin or Noggin. Development    124, 453-460.-   Fan, M. J., and Sokol, S. Y. (1997). A role for Siamois in Spemann    organizer formation. Development 124, 2581-2589.-   Fredieu, J. R., Cui, Y., Maier, D., Danilchik, M. V., and    Christian, J. L. (1997). XWnt-8 and lithium can act upon either    dorsal mesoderm or neurectodermal cells to cause a loss of forebrain    in Xenopus embryos. Developmental Biology 186, 100-114.-   Gavalas, A., and Krumlauf, R. (2000). Retinoid signalling and    hindbrain patterning [In Process Citation]. Curr Opin Genet Dev 10,    380-6.-   Glinka, A., Wu, W., Delius, H., Monaghan, A. P., Blumenstock, C.,    and Niehrs, C. (1998). Dickkopf-1 is a member of a new family of    secreted proteins and functions in head induction.-   Glinka, A., Wu, W., Onichtchouk, D., Blumenstock, C., and Niehrs, C.    (1997). Head induction by simultaneous repression of Bmp and Wnt    signalling in Xenopus. Nature 389, 517-519.-   Gould, A., Itasaki, N., and Krumlauf, R. (1998). Initiation of    rhombomeric Hoxb4 expression requires induction by somites and a    retinoid pathway. Neuron 21, 39-51.-   Grapin-Botton, a., Bonnin, M.-A., and Le Douarin, N. (1997). Hox    gene induction in the neural tube depends on three parameters:    competence, signal supply and paralogue group. Development 124,    849-859.-   Hamburger, V., and Hamilton, H. L. (1951). A series of normal stages    in the development of the chick embryo. J. Morph. 88, 49-92.-   He, X., Saint-Jeannet, J. P., Wang, Y., Nathans, J., Dawid, I., and    Varmus, H. (1997). A member of the Frizzled protein family mediating    axis induction by Wnt-5A. Science 275, 1652-4.-   Heasman, J., Kofron, M., and Wylie, C. (2000). ∃-catenin signaling    activity dissected in the early Xenopus embryo: a novel antisense    approach. Dev Biol 222, 124-34.-   Heisenberg, C. P., Tada, M., Rauch, G. J., Saude, L., Concha, M. L.,    Geisler, R., Stemple, D. L., Smith, J. C., and Wilson, S. W. (2000).    Silberblick/Wnt11 mediates convergent extension movements furing    zebrafish gastrulation. Nature 405, 76-81.-   Hemmati-Brivanlou, A., Kelly, O. G., and Melton, D. A. (1994).    Follistatin, an antagonist of activin, is expressed in the Spemann    organizer and displays direct neuralizing activity. Cell 77, 283-95.-   Hemmati-Brivanlou, A., and Melton, D. (1997). Vertebrate embryonic    cells will become nerve cells unless told otherWise. Cell 88, 13-7.-   Hemmati-Brivanlou, A., and Melton, D. a. (1994). Inhibition of    activin receptor signaling promotes neuralization in Xenopus. Cell    77, 273-81.-   Hoppler, S., Brown, J. D., and Moon, R. T. (1996). Expression of a    dominant-negative Wnt blocks induction of MyoD in Xenopus embryos.    Genes Dev. 10, 2805-2817.-   Hsieh, J.-C., Kodjabachian, L., Rebbert, M. L., Rattner, A.,    Smallwood, P. M., Samos, C. H., Nusse, R., Dawid, I. B., and    Nathans, J. (1999). A new secreted protein that binds to Wnt    proteins and inhibits their activities. Nature 398, 431-436.-   Itaskai, N., Sharpe, J., Morrison, A., and Krumlauf, R. (1996).    Reprogramming Hox expression in the vertebrate hindbrain: Influence    of paraxial mesoderm and rhombomere transposition. Neuron 16,    487-500.-   Itoh, K., and Sokol, S. (1997). Graded amounts of Xenopus    dishevelled specify discrete anteroposterior cell fates in    prospective ectoderm. Mechanism of Development 61, 113-125.-   Itoh, K., and Sokol, S. Y. (1999). Axis determination by inhibition    of Wnt signaling in Xenopus. Genes Dev 13, 2328-36.-   Itoh, K., Tang, T. L., Neel, B. G., and Sokol, S. Y. (1995).    Specific modulation of ectodermal cell fates in Xenopus embryos by    glycogen synthase kinase. Development 121, 3979-3988.-   Jones, C. M., and Smith, J. C. (1999). An overview of Xenopus    development. Methods in Molecular Biology 97, 331-340.-   Jones, C. M., and Smith, J. C. (1999). Wholemount in situ    hybridization to Xenopus embryos. Methods in Molecular Biology 97,    635-640.-   Joyner, A. L. (1996). Engrailed, Wnt and pax genes regulate    midbrain-hindbrain development. Trends Genet 12, 15-20.-   Kintner, C. (1992). Molecular bases of early neural development in    Xenopus embryos. Ann. Rev. Neurosci. 15, 251-284.-   Kolm, P., Apekin, V., and Sive, H. (1997). Xenopus hindbrain    patterning requires retinoid signaling. Developmental Biology 192,    1-16.-   Kreig, P., and Melton, D. (1988). In vitro RNA synthesis with SP6    RNA polymerase. In Methods in Enzymology: Recombinant DNA    techniques, S. Berger and A. Kimmel, eds.: Academic Press), pp.    397-415.-   Lamb, T. M., and Harland, R. M. (1995). Fibroblast growth factor is    a direct neural inducer, which combined with noggin generates    anterior-posterior neural pattern. Development 121, 3627-3636.-   Lee, K. J., and Jessell, T. M. (1999). The specification of dorsal    cell fates in the vertebrate central nervous system. Annu Rev    Neurosci 22, 261-94.-   Leyns, L., Bouwmeester, T., Kim, S. H., Piccolo, S., and    DeRobertis, E. M. (1997). Frzb-1 is a secreted antagonist of Wnt    signaling expressed in the Spemann organizer. Cell 88, 747-56.-   Liem, K. F., Jr., Tremml, G., and Jessell, T. M. 1997). A role for    the roof plate and its resident TGF ∃-related proteins in neuronal    patterning in the dorsal spinal cord. Cell 91, 127-38.-   Liem, K. F., Jr., Tremml, G., Roelink, H., and Jessell, T. M.    (1995). Dorsal differentiation of neural plate cells induced by    BMP-mediated signals from epidermal ectoderm. Cell 82, 969-79.-   Lin, X., and Perrimon, N. (1999). Dally cooperates with Drosophila    Frizzled2 to transduce Wingless signalling. Nature 400, 281-284.-   Lu, J., Chuong, C., and Widelitz, R. (1997). Isolation and    characterization of chicken ∃-catenin. Gene 196, 201-207.-   Lumsden, A., and Krumlauf, R. (1996). Patterning the vertebrate    neuraxis. Science 274, 1109-1115.-   McGrew, L. Hoppler, S., and Moon, R. (1997). Wnt and FGF pathways    cooperatively pattern anteroposterior neural ectoderm in Xenopus.    Mechanisms of Development 69, 105-114.-   McGrew, L., Lai, C.-J., and Moon, R. (1995). Specification of the    anteroposterior neural axis through synergistic interaction of the    Wnt signalling cascade with noggin and follistatin. Developmental    Biology 172, 337-342.-   McGrew, L., Takemaru, K., Bates, R., and Moon, R. (1999). Direct    regulation of the Xenopus engrailed-2 promoter by the Wnt signaling    pathway, and a molecular screen for Wnt-responsive genes, confirm a    role for Wnt signaling during neural patterning in Xenopus.    Mechanism of Development 87, 21-32.-   McMahon, A. P., Joyner, A. L., Bradley, A., and McMahon, J. A.    (1992). The midbrain-hindbrain phenotype of Wnt-1−/Wnt-1− mice    results from stepWise deletion of engrailed-expressing cells by 9.5    days postcoitum. Cell 69, 581-95.

Moon, R. T., Brown, J. D., Yang-Snyder, J. A., and Miller, J. R. (1997).Structurally related receptors and antagonists compete for secreted Wntligands. Cell 88, 725-8.

-   Muhr, J., Graziano, E., Wilson, S., Jessell, T., and Edlund, T.    (1999). Convergent inductive signals specify midbrain, hindbrain,    and spinal cord identity in gastrula stage chick embryos. Neuron 23,    689-702.-   Muhr, J., Jessell, T., and Edlund, T. (1997). Assignment of early    caudal identity to neural plate cells by a signal from caudal    paraxial mesoderm. Neuron 19, 487-502.-   Munsterberg. A. E., Kitajewski, J., Bumcrot, D. A., McMahon, A. P.,    and Lassar, A. B. (1995). Combinatorial signaling by Sonic hedgehog    and Wnt family members induces myogenic bHLH gene expression in the    somite. Genes Dev 9, 2911-22.-   Nieuwkoop, P. (1952). Activation and organisation of the central    nervous system in amphibians. J. Exp. Zool. 120, 1-108.-   Pera, E. M., and DeRobertis, E. M. (2000). A direct screen for    secreted proteins in Xenopus embryos identifies distinct activities    for the Wnt antagonists Crescent and Frzb-1. Mech Dev 96, 183-95.-   Piccolo, S., Agius, E., Leyns, L., and Bhattacharyya, S. (1999). The    head inducer Cerberus is a multifunctional antagonist of Notal, BMP    and Wnt signals. Nature 397, 707-710.-   Piccolo, S., Sasai, Y., Lu, B., and DeRobertis, E. M. (1996).    Dorsoventral patterning in Xenopus: inhibition of ventral signals by    direct binding of chordin to BMP-4. Cell 86, 589-98.-   Pinson, K. I., Brennan, J., Monkley, S., Avery, B. J., and    Skarnes, W. C. (2000). An LDL-receptor-related protein mediates Wnt    signalling in mice. Nature 407, 535-538.-   Pownall, M. E., Isaacs, H. V., and Slack, J. M. (1998). Two phases    of Hox gene regulation during early Xenopus development. Curr Biol    8, 673-6.-   Pownall, M. E., Tucker, A. S., Slack, J. M., and Isaacs, H. V.    (1996). eFGF, Xcad3 and Hox genes form a molecular pathway that    establishes the anteroposterior axis in Xenopus. Development 122,    3881-92.-   Rasmussen, J. T., Deardorff, M. A., Tan, C., Rao, M. S., Klein, P.    S., and Vetter, M. L. (2001). Regulation of eye development by    frizzled signaling in Xenopus. Proc Natl Acad Sci USA 98, 3861-6.-   Rothberg, J. M., Jacobs, J. R., Goodman, C. S., and    Artavanis-Tsakonas, S. (1990). slit: an extracellular protein    necessary for development of midline glia and commissural axon    pathways contains both EGF and LRR domains. Genes Dev 4, 2169-87.-   Ruiz i Altaba, A. (1994). Pattern formation in the vertebrate neural    plate. TINS 17, 233-243.-   Salic, A. N., Kroll, K. L., Evans, L. M., and Kirschner, M. W.    (1997). Sizzled: a secreted XWnt8 antagonist expressed in the    ventral marginal zone of Xenopus embryos. Development 124,    4739-4748.-   Tada, M., and Smith, J. (2000). XWnt11 is a target of Xenopus    Brachyury: regulation of gastrulation movements via Dishevelled, but    not through the canonical Wnt pathway. Development 127, 2227-2238.-   Tamai, K., Semenov, M., Kato, Y., Spokony, R., Lui, C., Katsuyama,    Y., Hess, F., Saint-Jeannet, J.-P., and He, X. (2000).    LDL-receptor-related proteins in Wnt signal transduction. Nature    407, 530-535.-   Trainor, P., and Krumlauf, R. (2000). Plasticity in mouse neural    crest cells reveals a new patterning role for cranial mesoderm.    Nature Cell Biology 2, 96-102.-   Tsuda, M., Kamimura, K., Nakato, H., Archer, M., Staatz, W., Fox,    B., Humphrey, M., Olson, S., Futch, T., Kaluza, V., Siegfried, E.,    Stam, L., and Selleck, S. B. (1999). The cell-surface protteoglycan    Dally regulates Wingless signalling in Drosophila. Nature 400,    276-280.-   Vleminckx, K., Kemler, R., and Hecht, A. (1999). The C-terminal    transactivation domain of ∃-catenin is necessary and sufficient for    signaling by the LEF-1/∃-catenin complex in Xenopus laevis. Mech Dev    81, 65-74.-   von Heijne, G. (1986). A new method for predicting signal sequence    cleavage sites. Nucleic Acid Research 14, 4683-4690.-   Wallingford, J. B. Rowning, B. A., Vogeli, K. M., Rothbacher, U.,    Fraser, S. E., and Harland, R. M. (2000). Dishevelled controls cell    polarity during Xenopus gastrulation. Nature 405, 81-5.-   Wang, S., Krinks, M., Lin, L., Luyten, F. P., and Moos, M., Jr.    (1997). Frzb, a secreted protein expressed in the Spemann organizer,    binds and inhibits Wnt-8. Cell 88, 757-66.-   Wehrli, N., Dougan, S. T., Caldwell, K., O'Keefe, L., Schwartz, S.,    Vaizel-Ohayon, D., Schejter, E., Tomlinson, A., and DiNardo, S.    (2000). Arrow encodes an LDL-receptor-related protein essential for    Wingless signalling. Nature 407, 527-530.

Thus, there has been shown and described novel methods and compositionsrelated to Wise, Sost, and LRP, which influence ocular development, bonedeposition, Wnt pathway, and tooth development, which fulfills all theobjects and advantages sought therefore. It is apparent to those skilledin the art, however, that many changes, variations, modifications, andother uses and applications for the subject methods and compositions arepossible, and also such changes, variations, modifications, and otheruses and applications which do not depart from the spirit and scope ofthe invention are deemed to be covered by the invention, which islimited only by the claims which follow.

1-181. (canceled)
 182. A method for affecting tooth developmentcomprising: (a) isolating Sost nucleic acid sequence; (b) attaching astop code at the beginning of the Sost nucleic acid sequence to form aSost cassette; (c) forming a Sost plasmid by inserting the Sost cassetteinto the plasmid; (d) transfecting a host cell with the Sost plasmid,whereby homologous recombination occurs with a wild type Sost gene; and(e) activating the stop code to cause a loss of function mutation.183-235. (canceled)